Synthesis and Structure–Activity Relationships of Pyrazolo [1, 5-a

Article pubs.acs.org/jmc

Synthesis and Structure−Activity Relationships of Pyrazolo[1,5a]pyridine Derivatives: Potent and Orally Active Antagonists of Corticotropin-Releasing Factor 1 Receptor Yoshinori Takahashi,*,† Shigeki Hibi,† Yorihisa Hoshino,† Koichi Kikuchi,† Kogyoku Shin,† Kaoru Murata-Tai,‡ Masae Fujisawa,§ Mitsuhiro Ino,∥ Hisashi Shibata,∥ and Masahiro Yonaga† †

Medicinal Chemistry, ∥Biopharmacology, ‡Physical Chemistry, and §Drug Metabolism and Pharmacokinetics, Tsukuba Research Laboratories, Eisai Co., Ltd., 5-1-3 Tokodai, Tsukuba-shi, Ibaraki 300-2635, Japan S Supporting Information *

ABSTRACT: Design, synthesis, and structure−activity relationships of a series of 3-dialkylamino-7phenyl pyrazolo[1,5-a]pyridines (I) as selective antagonists of the corticotropin-releasing factor 1 (CRF1) receptor are described. The most prominent compound to emerge from this work, 46 (E2508), exhibits potent in vitro activity, excellent drug-like properties, and robust oral efficacy in animal models of stress-related disorders. It has advanced into clinical trials.

■

INTRODUCTION Tricyclic antidepressants and serotonin and/or norepinephrine transporter reuptake inhibitors are widely used in the treatment of depression, anxiety, and stress-related disorders. These medicines, although clinically important, are not ideal owing to their insufficient efficacy, slow onset of action, or adverse events such as nausea and sexual dysfunction. Thus, new drugs with novel mechanisms of action, which may address some or all of the above unmet medical needs, have been long awaited in this area. Corticotropin-releasing factor (CRF), first isolated by Vale from ovine brain extract in 1981,1 is a 41-amino-acid neuropeptide secreted from the hypothalamus. It is closely linked to the release of adrenocorticotropic hormone from the pituitary and plays an important role in the regulation of the hypothalamic−pituitary−adrenal axis.2 CRF exerts its effects through two receptor subtypes (CRF1 and CRF2) that belong to the class B subfamily of G-protein-coupled receptors,3−6 which are widely distributed throughout the central and peripheral nervous systems. Preclinical and clinical evidence implicate CRF1 receptors in stress-related diseases. For instance, CRF1 receptor knockout mice show less anxious behavior,7,8 and intracerebroventricular administration of CRF to rats induces anxiety and depression-like behavioral changes.9 Moreover, in humans, high CRF levels in cerebrospinal fluid have been found in patients with depression.10,11 By contrast, the role of CRF2 as a target for stress-related disorders has not been fully established. Thus, it is hypothesized that selective CRF1 receptor antagonists would be useful in the treatment of stress-related diseases such as depression, anxiety, and, possibly, irritable bowel syndrome. Indeed, the efficacy of CRF1 receptor © 2012 American Chemical Society

antagonists has been widely shown in preclinical animal models of anxiety and depression.12−19 However, the clinical utility of CRF1 receptor antagonists has not yet been fully demonstrated. Compound 1 (R121919) exhibited antidepressant/anxiolytic activity in patients with depression in a small open-label phase IIa study,20,21 whereas 4 (CP-316,311) failed to show efficacy in a double-blind, placebocontrolled study22 (Figure 1). It is tempting to speculate that the main reason for these equivocal results lies with the compound itself (e.g., suboptimal drug-like properties such as high lipophilicity or low water solubility, and inadequate target engagement) and not with the mechanism of action. Therefore, we hypothesized that a CRF1 receptor antagonist having the appropriate drug-like characteristics may show robust efficacy as a novel antidepressant in clinical practice. Analyses of CRF1 receptor antagonists published so far, such as 1,23 2 (CP-154,526),24 3 (DMP696),25 and 4,26 indicate the following structural features:27 (1) a 6,5-, or 6,6-fused heteroaromatic core or monocyclic core containing an sp2hybridized nitrogen atom, which is thought to be essential as a hydrogen acceptor; (2) a small alkyl or tertiary amine group attached to the core ring; (3) an aryl (or heteroaryl) ring appended to the core and mono- or di-ortho-substituted to maintain an appropriate orthogonal conformation (Figure 2). According to the above-mentioned requirements, we focused on a 5,6-fused bicyclic heteroaromatic template (I) with both the sp2-hybridized nitrogen atom and the dialkylamino group on the core’s five-membered ring; this structural system is Received: February 24, 2012 Published: May 15, 2012 5255

dx.doi.org/10.1021/jm300259r | J. Med. Chem. 2012, 55, 5255−5269

Journal of Medicinal Chemistry

Article

Figure 1. Reported 6,5-fused or monocyclic CRF1 receptor antagonists.

2-bromopyridine 5 with 1-butyne afforded 6. N-Amination of 6 with O-mesitylenesulfonylhydroxylamine,28 followed by cyclization in the presence of sodium methoxide in methanol, generated the 2-ethyl pyrazolo[1,5-a]pyridine core 7 (Scheme 1). Bromination of 7 and the 2-methyl analogue 1129 gave 8 and 12, respectively. Subsequent nitration and reduction produced the corresponding amines, which were protected to give tert-butyl carbamates 10 and 14, respectively (Scheme 2). Schemes 3 and 4 show the syntheses of 2-methoxy- and 2methylthio-substituted core rings, respectively, using procedures similar to those in Scheme 2. Thus, 1530 was converted to tert-butyl carbamate 18 in three steps via nitrosation by using NaNO2/AcOH; note that NO2BF4 gave a complex reaction mixture, whereas Cu(NO3)2·3H2O failed to promote the reaction. Similar to the 2-methoxy intermediates, reduction of the nitro group of 1931 followed by conversion to tert-butyl carbamate gave 20, which was iodinated to 21. The preparation of pyrazolo[1,5-a]pyridine derivatives 24, 27, 30, and 33 is described in Schemes 5 and 6. Alkylation of 10, 18, 21, and 14 with alkyl bromides or alkyl tosylates in the presence of sodium hydride, followed by removal of the Boc group gave 22, 25, 28, and 31, respectively. Subsequent reductive amination afforded the corresponding tertiary amines. Suzuki coupling of the amines with various arylboronic acids gave the desired products (Scheme 5). Alternatively, the palladium-catalyzed reaction to introduce various aryl groups was carried out prior to elaboration of the dialkylamino moiety to afford the desired products 24, 27, and 30 (Scheme 6). Suzuki coupling was also investigated as a method for introduction of the aryl group into the nitro compound 9 (Scheme 7). Reduction of the nitro group of 40 followed by reductive amination yielded the symmetrical dialkyl amine 24. The synthesis of 2-cyano analogue 45 is shown in Scheme 8. Oxidation of the methylthio group in 19 gave the sulfone 41. Then, displacement of the methylsulfonyl group with cyano group gave 42. Finally, introduction of the aryl group via iodide 44 afforded the desired product 45.

Figure 2. Designed template I based on the structural similarity of CRF1 receptor antagonist.

unprecedented among CRF1 receptor antagonists described so far. In this paper, we report the synthesis, structure−activity relationships, and in vivo efficacy in animal models for stressrelated disorders for a series of pyrazolo[1,5-a]pyridine derivatives. Our research efforts in this area have led to the discovery of 46 as a promising CRF1 receptor antagonist that has advanced into clinical trials.

■

RESULTS AND DISCUSSION Chemistry. The syntheses of 7, 10, and 14, which are intermediates to 2-alkyl pyrazolo[1,5-a]pyridine analogues, are described in Schemes 1 and 2. Palladium-catalyzed coupling of Scheme 1a

a Reagents and conditions: (a) 1-Butyne, PdCl2(PPh3)2, cat. CuI, Et2NH, rt, 83%; (b) (i) O-mesitylenesulfonylhydroxylamine, CH2Cl2, 0 °C, (ii) MeONa, MeOH, 0 °C, 38%, 2 steps.

Scheme 2a

Reagents and conditions: (a) BrF2CCF2Br, n-BuLi in hexane, THF, −78 °C−rt, 84−86%; (b) NO2BF4, CH3CN, 0 °C, 57−60%; (c) (i) Zn, AcOH, EtOH, H2O, 60 °C, 47−60%, (ii) Boc2O, Et3N, CH2Cl2, 0 °C−rt, 79−83%.

a

5256

dx.doi.org/10.1021/jm300259r | J. Med. Chem. 2012, 55, 5255−5269

Journal of Medicinal Chemistry

Article

Scheme 3a

Reagents and conditions: (a) BrCl2CCCl2Br, n-BuLi in hexane, THF, −78 °C−rt, 65%; (b) NaNO2, AcOH, H2O, rt, then Zn, EtOH, H2O, 60 °C, 70%; (c) Boc2O, Et3N, CH2Cl2, rt, 97%.

a

Scheme 4a

Prediction of the metabolic pathways of 24d highlighted the alkylamine moiety at C-3 as a possible cause for the metabolic instability. Thus, the side chain was modified at the 3-position, as summarized in Table 2. In order to improve hCLint, introduction of polar groups at one of the N-substituents was explored while retaining the other cyclopropylmethyl group. Although introduction of methoxy (24h) and fluorine (24i) slightly reduced lipophilicity, these compounds showed higher hCLint than 24d. Subsequently, cyclic ether groups were introduced, based on the hypothesis that the cause of the metabolic instability might be O-demethylation in 24h. Unfortunately, 3- and 2-THF analogues, 24j and 24k, respectively, showed high hCLint. However, 4-tetrahydropyranyl (4-THP) analogue 24l displayed significant improvement in hCLint while maintaining potent binding affinity. Metabolism studies on these compounds showed mono- and didealkylation of the 3-amino side chain as prominent pathways; oxidation of the α-carbon to the oxygen atom on the cyclic ether could also contribute to metabolic instability. The improved stability of 24l relative to 24j/24k does not appear to stem from the overall lipophilicity of the molecule. We speculate that the position of the oxygen atom on the cyclic ether might contribute to reducing the metabolism at the α position of the THP ring.34 Since the 4-THP analogue 24l had improved hCLint, modification of its 7-aryl ring was examined to enhance solubility35 (Table 3). Replacement of the ortho methyl group with a methoxy group (24m) slightly improved solubility while maintaining comparable binding affinity and hCLint. However, regioisomer 24n did not have improved solubility. Since 24m showed higher in vitro affinity as well as better solubility than 24n, modification of the para position of the C-7 ortho dimethoxyphenyl ring was explored. While the solubility of the methoxy derivative 24o was almost identical to that of 24m, introduction of hydroxymethyl (24q) greatly improved the solubility but only provided moderate affinity. However, the methoxymethyl analogue 24r showed the appropriate balance

Reagents and conditions: (a) (i) Zn, AcOH, EtOH, H2O, 80 °C, (ii) Boc2O, Et3N, CH2Cl2, rt, 43%, 2 steps; (b) 1,2-diiodoethane, n-BuLi in hexane, THF, −78 °C, 69%. a

Pharmacology. The affinity for human CRF1 receptors was determined by competition with 125I-CRF using cell membranes prepared from human CRF1 receptor expressed in HEK293 cells; the functional antagonistic activities of CRFstimulated cAMP production were determined in HEK293 cells expressing the human CRF1 receptor.32 To evaluate the potential of the 5,6-fused heteroaromatic template, we first investigated pyrazolo[1,5-a]pyridine compounds 24a, 24b, 24f, and 24g, bearing 2,4-disubstituted phenyl rings at C-7 and dialkylamino groups at C-3 (Table 1). As can be seen, these pyrazolo[1,5-a]pyridine derivatives exhibited potent binding activity. In addition, 3-dicyclopropylmethylamino derivatives showed somewhat increased activity, especially functional antagonistic activity, when compared with di-n-propyl analogues. Other substitution patterns such as the methyl analogue 24c and the trisubstituted analogues 24d and 24e also displayed significant binding affinity. Regarding the functional antagonistic activities, trisubstituted analogue 24d, with an ortho methoxy group, tended to be more potent than the disubstituted analogue 24c. Unfortunately, 24d exhibited high intrinsic clearance (hCLint; 1.12 mL/min/mg) in human liver microsomes, probably because of its high lipophilicity (clogP33 = 6.8). Therefore, modification of 24d became the focus for improvement of the drug-like properties, especially human metabolic stability. Scheme 5a

a Reagents and conditions: (a) (i) R1-Br or R1-OTs, NaH, DMF, rt−40 °C, (ii) HCl in AcOEt, rt−40 °C, 77−100%, 2 steps; (b) Alkylaldehyde, NaBH(OAc)3, THF, rt, 58−99%; (c) Ar−B(OH)2, Pd(PPh3)4, Ba(OH)2·8H2O, DME, H2O, reflux, 37−98%, or Ar−B(OH)2, Pd(PPh3)4, K2CO3, DME, H2O, reflux, 61−93%.

5257

dx.doi.org/10.1021/jm300259r | J. Med. Chem. 2012, 55, 5255−5269

Journal of Medicinal Chemistry

Article

Scheme 6a

Reagents and conditions: (a) Ar−B(OH)2, Pd(PPh3)4, DME, H2O and Ba(OH)2·8H2O or K2CO3, 80−90 °C, 35−99%; (b) (i) R1-Br or R1-I or R1OMs, NaH, DMF, 40−50 °C, (ii) HCl in AcOEt, or TFA, CH2Cl2, rt−40 °C; (c) Alkylaldehyde, NaBH(OAc)3, THF; or NaBH(OAc)3, THF, AcOH; or NaBH4, 3 M H2SO4, THF; or α-picoline-borane, MeOH, rt, 16−86%, 3 steps; (c) for 30c (i) Alkylaldehyde, NaBH(OAc)3, THF, rt, (ii) TBAF, THF, rt, 19%, 2 steps; (c) for 30g (i) Alkylaldehyde, NaBH(OAc)3, THF, AcOH, rt, 43%, (ii) TBAF, THF, rt, 78%, (iii) MeI, NaH, DMF, rt, 94%. a

Scheme 7a

Table 1. Initial Investigation of Pyrazolo[1,5-a]pyridine Derivatives

a Reagents and conditions: (c) Ar−B(OH) 2, Pd(PPh3) 4, Ba(OH)2·8H2O, DME, H2O, 80 °C, 73−100%; (b) (i) Zn, AcOH, EtOH, H2O, 60 °C, (ii) Alkylaldehyde, NaBH4, 3 M H2SO4, THF, 0 °C−rt, 66−77%, 2 steps.

of solubility, potent activity, and improved hCLint. The fact that clogP is lower for 24r (4.73) than for 24m (5.43) might contribute to the improved hCLint. Regioisomer 24s also showed comparable solubility but less activity than 24r, indicating that bulky substituents might be less well tolerated at the ortho position than the para position. Compound 24t showed identical affinity to 24r, but somewhat lower solubility and increased hCLint. n-Propyl analogues 24v and 24w displayed 2-fold lower binding affinity and higher hCLint compared with 24m and 24r, respectively, suggesting that the cyclopropylmethyl group is preferable, in line with the results listed in Table 1. Next, the substituent effects at the 2-position of the promising compound 24r were examined (Table 4). The methoxy analogue 27a showed comparable binding affinity and

compd

R

binding IC50 (nM)a

cAMP IC50 (nM)a

24a 24b 24c 24d 24e 24f 24g

2-Cl-4-MeO 2-Me-4-MeO 2-MeO-4-Me 2-MeO-4,6-diMe 4-MeO-2,6-diMe 2-Cl-4-MeO 2-Me-4-MeO

10 16 12 24 13 15 57

100 150 100 59 90 200 480

a

All values are the averages of two measurements.

solubility, but displayed higher hCLint and weaker functional antagonism relative to 24r. However, introduction of methylthio (30a) preserved the binding affinity and hCLint, but decreased the solubility. The solubilities of 24r, 27a, and 30a were not correlated with the corresponding clogP values (4.7, 3.9, and 4.5, respectively). To confirm this, their relative

Scheme 8a

a Reagents and conditions: (a) alumina, oxone, H2O, CHCl3, 80 °C, 83%; (b) NaCN, THF, DMF, 80 °C, 69%; (c) (i) Pd/C, EtOAc, rt, 89%, (ii) cPrCHO, Ti(Oi-Pr)4, NaBH4, THF, MeOH, rt; (iii) 4-THP-CHO, α-picoline-borane, MeOH, AcOH, rt, 37%, 2 steps; (d) 1,2-diiodoethane, n-BuLi in hexane, THF, −78−40 °C, 63%; (e) Ar−B(OH)2, Pd(PPh3)4, 1 M Na2CO3, toluene, EtOH, 90 °C, 60%.

5258

dx.doi.org/10.1021/jm300259r | J. Med. Chem. 2012, 55, 5255−5269

Journal of Medicinal Chemistry

Article

Table 2. Effects of Dialkylamino Side Chain at the 3-Position of Pyrazolo[1,5-a]pyridine Derivatives

compd 24d 24h 24i 24j 24k 24l

R cPrCH2 MeOCH2CH2 FCH2CH2CH2 3-THFCH2 2-THFCH2 4-THPCH2

binding IC50 (nM)a

hCLint (mL/min/mg)b

24 41 20 38 34 22

1.12 1.69 1.41 1.35 1.23 0.45

Table 4. Effects of 2-Substituents in Pyrazolo[1,5-a]pyridine Derivatives

clogP 6.8 5.9 6.3 5.7 6.3 6.1

a

All values are the averages of two measurements. bRate of human intrinsic clearance in vitro. Each value represents the mean of six measurements.

R

241 24m 24n 24o 24p 24q

2-MeO-4,6-diMe 2,6-diMeO-4-Me 2,4-diMeO-6-Me 2,4,6-triMeO 2,4,5-triMeO 2,6-diMeO-4HOCH2 2,6-diMeO-4MeOCH2 2,4-diMeO-6MeOCH2 2,6-diMeO-4EtOCH2 2,6-diMeO 2,6-diMeO-4-Me 2,6-diMeO-4MeOCH2

24r 24s 24t 24u 24v 24w

binding IC50 (nM)a

solubility (μM)b

hCLint (mL/min/mg)c

22 22 40 42 130 200

0 1.6 0 2.5 3.6 30

0.45 0.44 0.45 0.45 0.60 NTd

50

4.8

0.30

120

2.9

0.53

49

1.4

0.52

105 40 90

3.3 1.3 3.1

0.74 0.89 0.70

Ar

X

24r 27a 30a 33 45 24m 27b 30b

1 1 1 1 1 2 2 2

Et OMe SMe Me CN Et OMe SMe

50 51 35 120 52 22 25 47

solubility (μM)b

hCLint (mL/min/mg)c

cAMP IC50 (nM)a

4.8 5.5 0 21 1.7 1.6 0.5 0

0.30 0.76 0.34 0.11 1.10 0.44 0.57 NTd

23 110 30 50 52 20 140 39

a All values are the averages of two measurements. bKinetic solubility in phosphate buffer saline using 10 mM DMSO stock solution. cRate of human intrinsic clearance in vitro. Each value represents the mean of six measurements. dNT = not tested.

Table 3. Effects of 7-Phenyl Substituents of Pyrazolo[1,5a]pyridine Derivatives

compd

compd

binding IC50 (nM)a

with a methyl group (33) decreased the binding affinity by more than 2-fold. Although introduction of a cyano group (45) as a polar substituent resulted in a binding affinity comparable to that of 24r, hCLint was poor. Modification of 24m to 2methoxy or 2-methylthio derivatives provided similar results to those for the methoxymethyl analogue 24r. Because compound 30a satisfied most of the targeted properties except solubility, modification of the 3-position was reinvestigated to explore the effects of additional polar groups on the solubility (Table 5). Introduction of OH (30c) or CN (30d), replacement of 4-THP with dioxane (30e, 30f), and modification of cyclopropylmethyl to methoxyethyl (30g) all improved the solubility, as expected. Among them, 30e and 30g showed potent binding affinity but weaker functional antagonism (cAMP IC50: 30e, 180 nM; 30g, 124 nM) compared with 30a (30 nM). This suggests that a polar group might be not functionally well tolerated at this position and that comparable lipophilicity to 24r might be necessary to acquire potent functional antagonistic activity. On balance, compound 24r was determined to be the most promising compound from this work and was subjected to further detailed investigations. The functional antagonism of 24r was also confirmed in a cAMP assay using the human neuroblastoma cell line IMR-32 expressing human CRF1 receptors (data not shown). Screening for salt and crystal forms using various acids such as HCl, H2SO4, MsOH, pTsOH, and HBr identified the pTsOH salt 46 (E2508) as the most promising crystalline form. The measured Ki values of 46 were 11 nM and >10 μM for hCRF1 and hCRF2 receptors, respectively. Compound 1, which was used as a positive control, displayed a Ki value of 8.3 nM in the assay.36 The anxiolytic efficacy of 46 was explored using the conventional light/dark test in mice (Figure 3).37 The compound significantly increased both the time spent in the light box and the number of crosses at 10 mg/kg p.o., suggesting that 46 may have the potential to ameliorate anxiety in patients.

a All values are the averages of two measurements. bKinetic solubility in phosphate buffer saline using 10 mM DMSO stock solution. cRate of human intrinsic clearance in vitro. Each value represents the mean of six measurements. dNT = not tested.

lipophilicities were measured by reversed-phase high-pressure liquid chromatography (HPLC) using an octadecylsilyl column; the retention times of 27a, 24r, and 30a were 85.9, 98.4, and 131.0, respectively, with reference to the t0 marker (NO3−). Thus, 30a is more lipophilic than 27a or 24r, in line with its reduced aqueous solubility. Replacement of the ethyl group 5259

dx.doi.org/10.1021/jm300259r | J. Med. Chem. 2012, 55, 5255−5269

Journal of Medicinal Chemistry

Article

3 mg/kg i.v. and 10 mg/kg p.o. (n = 4), respectively (Table 6). The results indicated a Tmax of 0.25 h, a half-life of 2.2 h, a

Table 5. Effects of 3-Dialkylamino Side Chain in 2Methylthio Derivatives

Table 6. Pharmacokinetic Parameters for 46 (i.v. and p.o.) in Male Micea i.v. (3 mg/kg) CL (L/h/kg) Vdss (L/kg) AUC(μg/mL·h) T1/2 (h) a

p.o. (10 mg/kg) 2.6 3.74 0.85 2.2

Cmax (μg/mL) Tmax (h) AUC(μg/mL·h) B.A. (%)

0.32 0.25 0.87 31

Each value represents the mean of four animals.

plasma clearance of 2.6 L/h/kg, and an oral bioavailability of 31%. The hepatic clearances calculated from in vitro metabolic parameters were comparable with in vivo total clearances measured in preclinical species. These results indicate that the main elimination route of 46 in animals is probably hepatic metabolism. Therefore, oral bioavailability in humans was predicted based on in vitro metabolic parameters using human liver microsomes. Moreover, 46 is not a substrate of human Pgp, and its offtarget liability panel screening did not show any significant affinity at various receptors and transporters up to 10 μM (data not shown). In addition, the inhibitory effect on the hERG channel was very weak (IC50 = 16.3 μM). Based on these data, 46 was selected as a clinical candidate.

■

a All values are the averages of two measurements. bKinetic solubility in phosphate buffer saline using 10 mM DMSO stock solution.

CONCLUSIONS We discovered a novel series of pyrazolo[1,5-a]pyridines as potent, drug-like CRF1 receptor antagonists. The combination of unique side-chains in the selected compound 46, namely, 4tetrahydropyranylmethyl and 2,6-dimethoxy-4-methoxymethylphenyl groups, afford improved human metabolic stability and solubility. 46 displays both high affinity and functional antagonism for the human CRF1 receptor (Ki = 11 nM), exhibits significant anxiolytic activity in a light/dark test in mice at 10 mg/kg (p.o.), and has the appropriate drug-like properties. Hence, it has advanced into clinical trials, where we believe it will prove the clinical usefulness of CRF1 receptor antagonists in the treatment of stress-related disorders such as depression and anxiety.

■

EXPERIMENTAL SECTION

Chemistry. 1H NMR spectra were recorded on a Bruker Avance spectrometer (operating at 600 MHz) or Varian Mercury 400 spectrometer (operating at 400 MHz). 13C NMR spectra were recorded on a Bruker Avance spectrometer (operating at 150 MHz) or JEOL JNM α400 spectrometer (operating at 100 MHz). Chemical shifts were calculated in ppm (δ) from the residual CHCl3 signal at (δH) 7.26 ppm and (δC) 77.0 ppm in CDCl3, or the residual C5HD4N signal at (δH) 8.71 ppm and (δC) 149.2 ppm or (δC) 123.5 ppm in C5D5N. High-resolution mass spectra (HRMS) were recorded on a ThermoFisherScientific LTQ-Orbitrap XL spectrometer (using electrospray ionization). For 46, the melting point was determined by visual inspection according to the U.S. Pharmacopeia. The infrared (IR) spectrum was obtained on a FT/IR 620 spectrometer (JASCO, JAPAN). The carbon, hydrogen, and nitrogen elemental concentrations were measured on a vario EL III (Elementar, USA). The purity of the biological tested compounds was determined by an analytical HPLC method and was found to be greater than or equal to 95% for all compounds. The parameters of the HPLC method were as follows: Accucore RP-MS column (2.1 × 50 mm, 2.6 μm); mobile phase: A = H2O with 0.1% HCO2H, B = acetonitrile with 0.1% HCO2H, 0−1 min, 0% B; 1−4 min, 0% B → 100% B; 4−8 min, 100%

Figure 3. Effects of 46 on mice in a light/dark test with 1 as a positive control. Each value represents the mean ± SEM of 24 mice. *p < 0.05 vs vehicle (Dunnett multiple comparison test).

The pharmacokinetic properties of 46 were evaluated in male ICR mice after intravenous and oral administration at doses of 5260

dx.doi.org/10.1021/jm300259r | J. Med. Chem. 2012, 55, 5255−5269

Journal of Medicinal Chemistry

Article

B; 8−11 min, 0% B; flow rate = 0.4 mL/min; detector: UV 254 nm; run time = 11 min. Reagents were purchased from commercial sources. Chromatography was performed on silica gel using the solvent systems indicated below. For mixed solvent systems, the volume ratios are given. 2-(1-Butynyl)pyridine (6). To a solution of 2-bromopyridine (504.6 g, 3.2 mol) in Et2NH (5 L) were added PdCl2(PPh3)2 (22.5 g, 32 mmol) and copper iodide (3.0 g, 16 mmol), and the reaction mixture was stirred for 10 h at room temperature while introducing 1butyne (400 g, 7.4 mol) as a gas. The reaction mixture was bubbled with nitrogen for 40 min, filtered through a pad of Celite to remove insoluble residue, and the filtrate washed with EtOAc. The organic extract was concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (n-hexane:EtOAc = 5:2) to afford 6 (380.5 g, 83%) as a brown oil. 1H NMR (400 MHz, CDCl3) δ 1.26 (t, J = 7.6 Hz, 3H), 2.45 (q, J = 7.6 Hz, 2H), 7.16−7.20 (m, 1H), 7.35−7.38 (m, 1H), 7.59−7.63 (m, 1H), 8.53−8.54 (m, 1H). 2-Ethylpyrazolo[1,5-a]pyridine (7). To a solution of 6 (260 g, 1.98 mol) in CH2Cl2 (1.6 L) was added a solution of Omesitylenesulfonylhydroxylamine (724 g, 2.6 mol, CAUTION!) in CH2Cl2 (900 mL) under ice cooling, and the reaction mixture was stirred for another one hour. Et2O (24 L) was added to the reaction mixture to precipitate crystals. The filtrate was filtered off and then dried under reduced pressure to afford a crude product of N-amino-2(1-butynyl)pyridinium mesitylenesulfonate as a pale yellow solid. To the obtained crude product in MeOH (600 mL) was added sodium methoxide in MeOH (28%, 309 mL, 1.6 mol) dropwise at 0 °C, and the mixture was vigorously stirred for 30 min. Ice−water (1.4 L) was added, and the solvent was evaporated under reduced pressure. The aqueous layer was extracted with EtOAc. The obtained organic extract was dried over MgSO4, filtered, and the solvent was distilled off under reduced pressure. The residue was purified by column chromatography on silica gel (n-hexane:EtOAc = 10:1) to afford 7 (88.2 g, 38%, 2 steps) as a light yellow oil. 1H NMR (400 MHz, CDCl3) δ 1.36 (t, J = 7.6 Hz, 3H), 2.86 (q, J = 7.6 Hz, 2H), 6.30 (s, 1H), 6.65 (ddd, J = 1.6, 6.8, 6.8 Hz, 1H), 7.04 (ddd, J = 1.2, 6.8, 8.8 Hz, 1H), 7.41 (ddd, J = 1.2, 1.2, 8.8 Hz, 1H), 8.37 (ddd, J = 1.2, 1.2, 6.8 Hz, 1H). 13C NMR (150 MHz, CDCl3) δ 157.8, 141.1, 128.2, 123.1, 117.3, 110.7, 94.5, 21.8, 13.9. HRMS calcd for (C9H10N2) [M + H]+ 147.0917; found 147.0919. 7-Bromo-2-ethylpyrazolo[1,5-a]pyridine (8). To a solution of 7 (25.6 g, 175 mmol) in THF (750 mL) was added n-BuLi (1.56 M hexane solution: 146 mL, 228 mmol) dropwise at −78 °C under a nitrogen atmosphere, and the reaction mixture was further stirred for 1 h at the same temperature. A solution of 1,2-dibromo-1,1,2,2tetrafluoroethane (59.2 g, 228 mmol) in THF (10 mL) was added dropwise to the reaction mixture, and stirred for 10 min at the same temperature. The mixture was warmed to 0 °C, and quenched carefully with a saturated NH4Cl aqueous solution at an internal temperature of −10 °C. The mixture was extracted with EtOAc, and the organic layer was washed with brine, dried over MgSO4, filtered, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (n-hexane:EtOAc = 11:1) to afford 8 (340 g, 86%) as a light yellow oil. 1H NMR (400 MHz, CDCl3) δ 1.36 (t, J = 7.6 Hz, 3H), 2.93 (q, J = 7.6 Hz, 2H), 6.49 (s, 1H), 6.94 (dd, J = 7.2, 8.4 Hz, 1H), 6.99 (dd, J = 1.6, 7.2 Hz, 1H), 7.44 (dd, J = 1.6, 8.4 Hz, 1H). 7-Bromo-2-ethyl-3-nitropyrazolo[1,5-a]pyridine (9). To a solution of 8 (102.9 g, 457.2 mmol) in CH3CN (880 mL) was added nitronium tetrafluoroborate (76.7 g, 548.6 mmol) under cooling with ice water, and the reaction mixture was stirred for 10 min. The reaction mixture was then poured into crashed ice, and was extracted with EtOAc. The organic layer was dried over MgSO4, filtered, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (n-hexane:EtOAc = 3:2) to give solid. The solid was washed with n-hexane to afford 9 (74.6 g, 60%) as a pale green solid. 1H NMR (400 MHz, CDCl3) δ 1.42 (t, J = 7.6 Hz, 3H), 3.27 (q, J = 7.6 Hz, 2H), 7.39 (dd, J = 1.2, 7.6 Hz, 1H), 7.50 (dd, J = 7.6, 8.8 Hz, 1H), 8.38 (dd, J = 1.2, 8.8 Hz, 1H). tert-Butyl N-(7-bromo-2-ethylpyrazolo[1,5-a]pyridin-3-yl)carbamate (10). To a suspension of 9 (74.6 g, 276 mmol) in

EtOH (1.3 L), water (650 mL), and AcOH (65 mL) was added zinc powder (75 g, 1.09 mol) at room temperature and the reaction mixture was stirred for 20 min at 60 °C. The reaction mixture was cooled and filtered through a pad of Celite to remove insoluble residue, and the filtrate was washed with EtOAc. The solvent was distilled off under reduced pressure, and the aqueous layer was extracted with EtOAc. The organic layer was washed with brine, dried over MgSO4, and filtered, and the solvent was distilled off under reduced pressure. The residue was purified by column chromatography on silica gel (n-hexane:EtOAc = 4:1) to give 7-bromo-2ethylpyrazolo[1,5-a]pyridine-3-amine (39.6 g, 60%) as a dark-brown solid. To a solution of 7-bromo-2-ethylpyrazolo[1,5-a]pyridine-3-amine (39.6 g, 165 mmol) from the previous step in CH2Cl2 (330 mL) was added Et3N (34.6 mL, 248 mmol), followed by Boc2O (45.5 mL, 198 mmol) under ice cooling, and the reaction mixture was stirred for 16 h. To the reaction mixture were added a saturated NaHCO3 aqueous solution and EtOAc. The mixture was filtered through a pad of Celite to remove insoluble residue, and the filtrate was washed with EtOAc. The solvent was evaporated, and the residue was extracted with EtOAc. The organic layer was washed with brine, dried over MgSO4, filtered, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (n-hexane:EtOAc = 4:1) to afford 10 (46.8 g, 83%) as a white solid. 1H NMR (400 MHz, CDCl3) δ 1.34 (t, J = 7.6 Hz, 3H), 1.52 (br s, 9H), 2.87 (q, J = 7.6 Hz, 2H), 5.91 (br s, 1H), 6.92−7.04 (m, 2H), 7.40 (d, J = 9.0 Hz, 1H). 13C NMR (150 MHz, CDCl3) δ 154.4, 153.3, 138.2, 123.4, 118.7, 115.3, 115.1, 108.0, 80.5, 28.3, 19.6, 13.4. HRMS calcd for (C14H18BrN3O2) [M + H]+ 340.0655; found 340.0660. tert-Butyl [2-(methylthio)pyrazolo[1,5-a]pyridin-3-yl]carbamate (20). Compound 20 was prepared according to the procedure described for the synthesis of 10 using 2-methylthio-3nitropyrazolo[1,5-a]pyridine 19 (400 mg, 1.9 mmol), zinc powder (800 mg, 12.2 mmol) in EtOH (20 mL), water (10 mL), AcOH (2 mL), Boc2O (625 mg, 2.9 mmol), and Et3N (0.40 mL, 2.9 mmol) in CH2Cl2 (5 mL). The product was purified by column chromatography on silica gel (n-hexane:EtOAc = 5:1) to afford 20 (230 mg, 43%, 2 steps) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ 1.53 (br s, 9H), 2.60 (s, 3H), 6.00−6.15 (m, 1H), 6.69 (t, J = 6.8 Hz, 1H), 7.11 (t, J = 8.0 Hz, 1H), 7.40−7.50 (m, 1H), 8.83 (d, J = 6.8 Hz, 1H). tert-Butyl [7-iodo-2-(methylthio)pyrazolo[1,5-a]pyridin-3yl]carbamate (21). Compound 21 was prepared according to the procedure described for the synthesis of 8 using 20 (21.6 g, 77.3 mmol) in THF (1 L), 1,2-diiodoethane (24.0 g, 85.0 mmol) in THF (50 mL) and n-BuLi (1.6 M hexane solution; 130 mL, 208.0 mmol). The product was purified by column chromatography on silica gel (nhexane:EtOAc = 5:1) to afford 21 (21.5 g, 69%) as a light yellow solid. 1 H NMR (400 MHz, CDCl3) δ 1.52 (s, 9H), 2.64 (s, 3H), 6.02−6.10 (m, 1H), 6.81 (dd, J = 7.2, 8.8 Hz, 1H), 7.22 (dd, J = 1.2, 7.2 Hz, 1H), 7.42−7.50 (m, 1H). 13C NMR (150 MHz, CDCl3) δ 153.9, 145.3, 136.7, 123.7, 123.1, 116.5, 110.4, 91.9, 80.8, 28.2, 15.5. HRMS calcd for (C13H16IN3O2S) [M + H]+ 406.0081; found 406.0085. 7-Bromo-N-(cyclopropylmethyl)-2-ethylpyrazolo[1,5-a]pyridin-3-amine (22a). To a solution of 10 (46.8 g, 138 mmol) in DMF (1 L) was added NaH (60%, 6.6 g, 179 mmol) at room temperature under a nitrogen stream, and the reaction mixture was stirred for 20 min. (Bromomethyl)cyclopropane (22.4 g, 166 mmol) was added at the same temperature, and the reaction mixture was stirred for 1 h at 40 °C. To the reaction mixture was gradually added ice. The mixture was extracted with EtOAc, and the organic layers was washed with brine, dried over MgSO4, filtered, and concentrated in vacuo to afford a crude product, which was used in the next step without purification. The crude product from the previous step was dissolved in 4 M HCl/EtOAc solution (500 mL) at room temperature, and the mixture was stirred for 30 min at 40 °C. 5 M NaOH aqueous solution was added to the reaction mixture at room temperature, and the residue was extracted with EtOAc. The organic layer was washed with brine, dried over MgSO4, filtered, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (n-hexane:EtOAc = 5261

dx.doi.org/10.1021/jm300259r | J. Med. Chem. 2012, 55, 5255−5269

Journal of Medicinal Chemistry

Article

3:1) to afford 22a (40.2 g, 99%, 2 steps) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ 0.14−0.22 (m, 2H), 0.47−0.56 (m, 2H), 0.96−1.10 (m, 1H), 1.37 (t, J = 7.6 Hz, 3H), 2.88 (d, J = 6.8 Hz, 2H), 2.90 (q, J = 7.6 Hz, 2H), 6.83 (dd, J = 7.0, 8.8 Hz, 1H), 6.90 (dd, J = 1.3, 7.1 Hz, 1H), 7.43 (dd, J = 1.3, 8.8 Hz, 1H). 13C NMR (150 MHz, CDCl3) δ 150.5, 135.8, 121.2, 120.6, 118.4, 115.0, 114.4, 56.0, 19.4, 14.0, 12.0, 3.4. HRMS calcd for (C13H16BrN3) [M + H]+ 294.0600; found 294.0604. 7-Bromo-N-(cyclopropylmethyl)-2-ethyl-N-[(tetrahydro-2Hpyran-4-yl)methyl]pyrazolo[1,5-a]pyridin-3-amine (23a). To a solution of 22a (40.2 g, 136.6 mmol) in THF (680 mL) was added tetrahydro-2H-4-pyrancarbaldehyde (31.2 g, 273.2 mmol) in THF (30 mL), followed by NaBH(OAc)3 (40.5 g, 191.2 mmol), and the mixture was stirred at room temperature for 30 min. To the reaction mixture was added ice, and the residue was extracted with EtOAc. The organic layer was washed with brine, dried over MgSO4, filtered, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (n-hexane:EtOAc = 5:1) to afford 23a (51.5 g, 96%) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ −0.02− 0.06 (m, 2H), 0.33−0.43 (m, 2H), 0.75−0.88 (m, 1H), 1.20−1.34 (m, 2H), 1.38 (t, J = 7.6 Hz, 3H), 1.48−1.62 (m, 1H), 1.69−1.78 (m, 2H), 2.88 (d, J = 6.8 Hz, 2H), 2.91 (q, J = 7.6 Hz, 2H), 3.04 (d, J = 7.0 Hz, 2H), 3.30 (dt, J = 2.1, 12.0 Hz, 2H), 3.90−4.00 (m, 2H), 6.88 (dd, J = 7.1, 8.8 Hz, 1H), 6.96 (dd, J = 1.3, 7.1 Hz, 1H), 7.49 (dd, J = 1.3, 8.8 Hz, 1H). 155.0, 138.9, 122.6, 122.0, 119.1, 115.7, 114.6, 67.9, 62.0, 61.8, 34.4, 31.6, 20.0, 14.0, 10.4, 3.7. HRMS calcd for (C19H26BrN3O) [M + H]+ 392.1332; found 392.1336. 7-Bromo-N,N-bis(cyclopropylmethyl)-2-ethylpyrazolo[1,5a]pyridin-3-amine (23e). Compound 23e was prepared according to the procedure described for the synthesis of 23a using 22a (150 mg, 0.51 mmol), cyclopropanecarboxaldehyde (76 μL, 1.0 mmol), and NaBH(OAc)3 (216 mg, 1.0 mmol) in THF (1.7 mL). The product was purified by column chromatography on silica gel (nheptane:EtOAc = 25:2) to afford 23e (166 mg, 93%) as a pale yellow oil. 1H NMR (600 MHz, CDCl3) δ −0.04−0.07 (m, 4H), 0.28−0.40 (m, 4H), 0.72−0.86 (m, 2H), 1.37 (t, J = 7.6 Hz, 3H), 2.92 (q, J = 7.7 Hz, 2H), 2.92−3.02 (m, 4H), 6.83 (dd, J = 7.2, 8.7 Hz, 1H), 6.91 (d, J = 7.2 Hz, 1H), 7.49 (d, J = 8.7 Hz, 1H). 13C NMR (150 MHz, CDCl3) δ 155.5, 139.0, 122.6, 121.7, 119.0, 115.9, 114.4, 60.6, 19.9, 14.0, 10.5, 3.6. HRMS calcd for (C17H22BrN3) [M + H]+ 348.1070; found 348.1070. 7-(2-Chloro-4-methoxyphenyl)-N,N-bis(cyclopropylmethyl)2-ethylpyrazolo[1,5-a]pyridin-3-amine (24a). To a suspension of 40a (115 mg, 0.35 mmol) in EtOH (5 mL), water (5 mL), and AcOH (0.1 mL) was added zinc powder (115 mg, 1.67 mol) at room temperature, and the reaction mixture was stirred for 30 min at 60 °C. The reaction mixture was cooled and filtered through a pad of Celite to remove insoluble residue, and the filtrate was washed with EtOAc. The solvent was evaporated under reduced pressure, and transferred to a separatory funnel. The aqueous layer was extracted with EtOAc. The organic layers were washed with brine, dried over MgSO4, filtered, and the solvent was concentrated under reduced pressure to give crude product, which was used in the next step without purification. The crude product from the previous step was dissolved in THF (8 mL). To this mixture was added cyclopropylmethyl carboxaldehyde (0.13 mL, 1.74 mmol) and 3 M H2SO4 aqueous solution (0.58 mL, 1.74 mmol) followed by NaBH4 (53 mg, 1.39 mmol) in five portions while vigorously stirring on ice, and stirring was continued for 20 min. Water was added to the reaction mixture, and the residue was transferred to a separatory funnel and was extracted with EtOAc. The combined organic layers were washed with brine, dried over MgSO4, filtered, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (n-hexane:EtOAc = 20:1) to afford 24a (94 mg, 66%, 2 steps) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ −0.02−0.06 (m, 4H), 0.30−0.38 (m, 4H), 0.77−0.90 (m, 2H), 1.26 (t, J = 7.5 Hz, 3H), 2.80 (q, J = 7.5 Hz, 2H), 2.99 (d, J = 6.6 Hz, 4H), 3.86 (s, 3H), 6,55 (dd, J = 1.3, 6.8 Hz, 1H), 6.92 (dd, J = 2.6, 8.6 Hz, 1H), 6.99 (dd, J = 6.8, 9.0 Hz, 1H), 7.06 (d, J = 2.4 Hz, 1H), 7.47 (d, J = 8.4 Hz, 1H), 7.49 (dd, J = 1.3, 8.8 Hz, 1H). 13C NMR (150 MHz, CDCl3) δ 160.6, 154.5, 137.5, 135.1, 132.3, 125.7, 121.1, 120.9, 116.4,

115.3, 112.8, 112.2, 60.7, 55.6, 19.8, 14.2, 10.6, 3.6. HRMS calcd for (C24H28ClN3O) [M + H]+ 410.1994; found 410.1997. N,N-Bis(cyclopropylmethyl)-2-ethyl-7-(4-methoxy-2methylphenyl)pyrazolo[1,5-a]pyridin-3-amine (24b). Compound 24b was prepared according to the procedure described for the synthesis of 24r using 23e (27 mg, 0.078 mmol), (4-methoxy-2methylphenyl)boronic acid (26 mg, 0.16 mmol), Pd(PPh3)4 (15 mg, 0.013 mmol), and Ba(OH)2·8H2O (49 mg, 0.16 mmol) in DME (0.6 mL) and water (0.3 mL). The product was purified by column chromatography on silica gel (n-heptane:EtOAc = 11:1) to afford 24b (25 mg, 81%) as a brown oil. 1H NMR (400 MHz, CDCl3) δ −0.02− 0.06 (m, 4H), 0.30−0.38 (m, 4H), 0.78−0.90 (m, 2H), 1.26 (t, J = 7.5 Hz, 3H), 2.08 (s, 3H), 2.81 (q, J = 7.5 Hz, 2H), 3.00 (d, J = 6.6 Hz, 4H), 3.86 (s, 3H), 6,49 (dd, J = 1.4, 6.7 Hz, 1H), 6.80−6.90 (m, 2H), 6.99 (dd, J = 6.8, 9.0 Hz, 1H), 7.34 (d, J = 8.2 Hz, 1H), 7.48 (dd, J = 1.4, 8.9 Hz, 1H). 13C NMR (150 MHz, CDCl3) δ 160.1, 154.7, 140.6, 139.7, 137.5, 131.1, 126.8, 121.1, 120.8, 115.7, 115.6, 111.6, 111.0, 60.7, 55.2, 19.9, 19.7, 14.3, 10.6, 3.6. HRMS calcd for (C25H31N3O) [M + H]+ 390.2540; found 390.2541. N,N-Bis(cyclopropylmethyl)-2-ethyl-7-(2-methoxy-4methylphenyl)pyrazolo[1,5-a]pyridin-3-amine (24c). Compound 24c was prepared according to the procedure described for the synthesis of 24a using 40b (70 mg, 0.22 mmol), zinc powder (70 mg, 1.1 mol) in EtOH (6 mL), water (3 mL), AcOH (1 mL), and cyclopropylmethyl carboxaldehyde (63 μL, 0.84 mmol), 3 M H2SO4 aqueous solution (0.28 mL, 0.84 mmol), NaBH4 (21 mg, 0.56 mmol) in THF (3 mL). The product was purified by column chromatography on silica gel (n-hexane:EtOAc = 10:1) to afford 24c (58 mg, 68%, 2 steps) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ 0.00−0.05 (m, 4H), 0.32−0.38 (m, 4H), 0.80−0.90 (m, 2H), 1.26 (t, J = 7.6 Hz, 3H), 2.43 (s, 3H), 2.80 (q, J = 7.6 Hz, 2H), 2.99 (d, J = 6.8 Hz, 4H), 3.75 (s, 3H), 6.59 (dd, J = 0.8, 6.8 Hz, 1H), 6.86 (s, 1H), 6.89 (br d, J = 7.6 Hz, 1H), 6.98 (dd, J = 6.8, 8.8 Hz, 1H), 7.41 (d, J = 7.6 Hz, 1H), 7.45 (dd, J = 0.8, 8.8 Hz, 1H). 13C NMR (150 MHz, CDCl3) δ 157.6, 154.0, 140.8, 137.9, 137.5, 130.9, 121.3, 121.0, 120.7, 120.7, 115.7, 112.7, 111.8, 60.7, 55.7, 21.8, 19.8, 14.2, 10.7, 3.6. HRMS calcd for (C25H31N3O) [M + H]+ 390.2540; found 390.2544. N,N-Bis(cyclopropylmethyl)-2-ethyl-7-(2-methoxy-4,6dimethylphenyl)pyrazolo[1,5-a]pyridin-3-amine (24d). Compound 24d was prepared according to the procedure described for the synthesis of 24h using 23e (40 mg, 0.12 mmol), (2-methoxy-4,6dimethyllphenyl)boronic acid (42 mg, 0.23 mmol), K2CO3 (48 mg, 0.35 mmol), and Pd(PPh3)4 (20 mg, 0.017 mmol) in DME (1 mL) and water (0.5 mL). The product was purified by column chromatography on silica gel (n-heptane:EtOAc = 10:1) to afford 24d (43 mg, 93%) as a pale yellow oil. 1H NMR (400 MHz, CDCl3) δ −0.02−0.04 (m, 4H), 0.30−0.36 (m, 4H), 0.80−0.90 (m, 2H), 1.22 (t, J = 7.6 Hz, 4H), 1.97 (s, 3H), 2.39 (s, 3H), 2.72−2.84 (m, 2H), 2.99 (d, J = 6.4 Hz, 2H), 3.68 (s, 3H), 6.48 (dd, J = 1.2, 6.8 Hz, 1H), 6.69 (s, 1H), 6.77 (s, 1H), 6.99 (dd, J = 6.8, 8.8 Hz, 1H), 7.46 (dd, J = 1.2, 8.8 Hz, 1H). 13C NMR (150 MHz, CDCl3) δ 157.6, 154.3, 139.8, 139.0, 137.5, 136.7, 123.4, 120.9, 120.5, 120.5, 115.5, 112.3, 110.0, 60.7, 55.9, 21.8, 19.7, 19.2, 14.5, 10.7, 3.6, 3.5. HRMS calcd for (C26H33N3O) [M + H]+ 404.2696; found 404.2701. N,N-Bis(cyclopropylmethyl)-2-ethyl-7-(4-methoxy-2,6dimethylphenyl)pyrazolo[1,5-a]pyridin-3-amine (24e). Compound 24e was prepared according to the procedure described for the synthesis of 24r using 23e (40 mg, 0.12 mmol), (2,6-dimethyl-4methoxyphenyl)boronic acid (42 mg, 0.23 mmol), Pd(PPh3)4 (20 mg, 0.017 mmol) and Ba(OH)2·8H2O (73 mg, 0.23 mmol) in DME (1 mL) and water (0.5 mL). The product was purified by column chromatography on silica gel (n-heptane:EtOAc = 13:1) to afford 24e (35 mg, 76%) as a yellow solid. 1H NMR (400 MHz, CDCl3) δ −0.02−0.06 (m, 4H), 0.30−0.40 (m, 4H), 0.81−0.94 (m, 2H), 1.24 (t, J = 7.5 Hz, 3H), 2.04 (s, 6H), 2.81 (q, J = 7.5 Hz, 2H), 3.03 (d, J = 6.6 Hz, 4H), 3.87 (s, 3H), 6,45 (dd, J = 1.5, 6.8 Hz, 1H), 6.73 (s, 2H), 7.02 (dd, J = 6.8, 8.8 Hz, 1H), 7.49 (dd, J = 1.4, 8.9 Hz, 1H). 13C NMR (150 MHz, CDCl3) δ 159.6, 154.8, 139.1, 138.9, 137.5, 126.5, 121.0, 120.6, 115.5, 112.9, 111.9, 60.7, 55.1, 19.9, 19.6, 14.5, 10.6, 3.5. HRMS calcd for (C26H33N3O) [M + H]+ 404.2696; found 404.2699. 5262

dx.doi.org/10.1021/jm300259r | J. Med. Chem. 2012, 55, 5255−5269

Journal of Medicinal Chemistry

Article

7-(2-Chloro-4-methoxyphenyl)-2-ethyl-N,Ndipropylpyrazolo[1,5-a]pyridin-3-amine (24f). Compound 24f was prepared according to the procedure described for the synthesis of 24a using 40a (90 mg, 0.27 mmol), zinc powder (180 mg, 2.8 mol) in EtOH (10 mL), water (5 mL), AcOH (2 mL), and propionaldehyde (58 μL, 0.81 mmol), 3 M H2SO4 aqueous solution (0.27 mL, 0.81 mmol), NaBH4 (20 mg, 0.54 mmol) in THF (2 mL). The product was purified by column chromatography on silica gel (n-hexane:EtOAc = 50:1) to afford 24f (70 mg, 67%, 2 steps) as a pale yellow oil. 1H NMR (400 MHz, CDCl3) δ 0.88 (t, J = 7.6 Hz, 6H), 1.23 (t, J = 7.6 Hz, 3H), 1.34−1.44 (m, 4H), 2.76 (q, J = 7.6 Hz, 2H), 3.02 (t, J = 7.6 Hz, 4H), 3.87 (s, 3H), 6.56 (dd, J = 1.6, 6.4 Hz, 1H), 6.92 (dd, J = 2.8, 8.6 Hz, 1H), 6.99 (dd, J = 6.4, 8.6 Hz, 1H), 7.06 (d, J = 2.8 Hz, 1H), 7.32 (d, J = 8.6 Hz, 1H), 7.45 (dd, J = 1.6, 8.6 Hz, 1H). 13C NMR (150 MHz, CDCl3) δ 160.6, 154.3, 137.6, 137.4, 135.0, 132.4, 125.6, 120.9, 120.6, 116.2, 115.3, 112.8, 112.4, 58.2, 55.6, 22.1, 19.9, 14.3, 11.7. HRMS calcd for (C23H28ClN3O) [M + H]+ 386.1994; found 386.1992. 2-Ethyl-7-(4-methoxy-2-methylphenyl)-N,Ndipropylpyrazolo[1,5-a]pyridin-3-amine (24g). Compound 24g was prepared according to the procedure described for the synthesis of 24r using 23g (40 mg, 0.12 mmol), (4-methoxy-2-methylphenyl)boronic acid (41 mg, 0.25 mmol), Pd(PPh3)4 (21 mg, 0.018 mmol), and Ba(OH)2·8H2O (78 mg, 0.25 mmol) in DME (1 mL) and water (0.5 mL). The product was purified by column chromatography on silica gel (n-hexane:EtOAc = 16:1) to afford 24g (40 mg, 89%) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ 0.88 (t, J = 7.6 Hz, 6H), 1.23 (t, J = 7.6 Hz, 3H), 1.34−1.44 (m, 4H), 2.10 (s, 3H), 2.75 (q, J = 7.6 Hz, 2H), 3.01 (t, J = 7.6 Hz, 4H), 3.86 (s, 3H), 6.49 (dd, J = 1.6, 6.8 Hz, 1H), 6.82 (dd, J = 2.8, 8.4 Hz, 1H), 6.86 (d, J = 2.8 Hz, 1H), 6.99 (dd, J = 6.8, 8.8 Hz, 1H), 7.32 (d, J = 8.4 Hz, 1H), 7.45 (dd, J = 1.6, 8.8 Hz, 1H). 13C NMR (150 MHz, CDCl3) δ 160.1, 154.5, 140.5, 139.7, 137.6, 131.2, 126.8, 121.2, 120.3, 115.6, 115.5, 111.8, 111.0, 58.3, 55.2, 22.1, 20.0, 19.8, 14.3, 11.7. HRMS calcd for (C26H33N3O2) [M + H]+ 366.2540; found 366.2538. N-(Cyclopropylmethyl)-2-ethyl-7-(2-methoxy-4,6-dimethylphenyl)-N-(2-methoxyethyl)pyrazolo[1,5-a]pyridin-3-amine (24h). To a sulution of 23b (40 mg, 0.11 mmol) in DME (1 mL) and water (0.5 mL) were added (2-methoxy-4,6-dimethylphenyl)boronic acid (41 mg, 0.23 mmol), K2CO3 (47 mg, 0.34 mmol), and Pd(PPh3)4 (20 mg, 0.017 mmol). The mixture was stirred at 90 °C for 14 h under a nitrogen stream and was then cooled and diluted with water. The mixture was extracted with EtOAc, and the organic layer was washed with brine, dried over MgSO4, filtered, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (nheptane:EtOAc = 7:1) to afford 24h (36 mg, 78%) as a pale yellow oil. 1 H NMR (600 MHz, CDCl3) δ −0.07−0.05 (m, 2H), 0.28−0.40 (m, 2H), 0.79−0.94 (m, 1H), 1.16−1.25 (m, 3H), 1.97 (s, 3H), 2.39 (s, 3H), 2.68−2.85 (m, 2H), 2.94−3.05 (m, 2H), 3.29 (s, 3H), 3.33−3.43 (m, 4H), 3.68 (s, 3H), 6.50 (d, J = 6.8 Hz, 1H), 6.69 (s, 1H), 6.76 (s, 1H), 7.01 (dd, J = 6.8, 8.7 Hz, 1H), 7.45 (d, J = 8.7 Hz, 1H). 13C NMR (150 MHz, CDCl3) δ 157.6, 154.3, 139.9, 139.0, 137.6, 136.7, 123.4, 121.3, 120.3, 119.6, 115.2, 112.6, 109.9, 71.9, 61.9, 58.7, 55.9, 54.8, 21.8, 19.5, 19.3, 14.4, 10.6, 3.5, 3.4. HRMS calcd for (C25H33N3O2) [M + H]+ 408.2646; found 408.2640. N-(Cyclopropylmethyl)-2-ethyl-N-(3-fluoropropyl)-7-(2-methoxy-4,6-dimethylphenyl)pyrazolo[1,5-a]pyridin-3-amine (24i). Compound 24i was prepared according to the procedure described for the synthesis of 24h using 23c (40 mg, 0.11 mmol), (2methoxy-4,6-dimethyllphenyl)boronic acid (40 mg, 0.22 mmol), K2CO3 (47 mg, 0.34 mmol), and Pd(PPh3)4 (20 mg, 0.017 mmol) in DME (1 mL) and water (0.5 mL). The product was purified by column chromatography on silica gel (n-heptane:EtOAc = 11:1) to afford 24i (42 mg, 91%) as a pale yellow oil. 1H NMR (400 MHz, CDCl3) δ −0.02−0.02 (m, 2H), 0.32−0.38 (m, 2H), 0.80−0.90 (m, 2H), 1.20 (t, J = 7.6 Hz, 3H), 1.70−1.82 (m, 2H), 1.97 (s, 3H), 2.39 (s, 3H), 2.74 (q, J = 7.6 Hz, 2H), 2.92 (d, J = 6.8 Hz, 2H), 3.30 (t, J = 6.8 Hz, 2H), 3.68 (s, 3H), 4.55 (td, J = 6.0, 47.2 Hz, 2H), 6.51 (dd, J = 1.2, 6.8 Hz, 1H), 6.69 (s, 1H). 6.77 (s, 1H), 7.02 (dd, J = 6.8, 8.8 Hz, 1H), 7.44 (dd, J = 1.2, 8.8 Hz, 1H). 13C NMR (150 MHz, CDCl3) δ 157.6, 154.1, 139.9, 139.0, 137.5, 136.8, 123.4, 121.4, 120.3, 119.3,

115.1, 112.6, 109.9, 82.5 (d, J = 163.7 Hz), 61.8, 55.9, 50.9 (d, J = 5.5 Hz), 30.0 (d, J = 18.8 Hz), 21.8, 19.7, 19.3, 14.4, 10.5, 3.6, 3.5. HRMS calcd for (C25H32FN3O) [M + H]+ 410.2602; found 410.2600. N-(Cyclopropylmethyl)-2-ethyl-7-(2-methoxy-4,6-dimethylphenyl)-N-[(tetrahydrofuran-3-yl)methyl]pyrazolo[1,5-a]pyridin-3-amine (24j). Compound 24j was prepared according to the procedure described for the synthesis of 24h using 23f (40 mg, 0.11 mmol), (2-methoxy-4,6-dimethyllphenyl)boronic acid (39 mg, 0.22 mmol), K2CO3 (44 mg, 0.32 mmol) and Pd(PPh3)4 (18 mg, 0.016 mmol) in DME (1 mL) and water (0.5 mL). The product was purified by column chromatography on silica gel (n-heptane:EtOAc = 5:1) to afford 24j (42 mg, 92%) as a pale yellow oil. 1H NMR (400 MHz, CDCl3) δ −0.02−0.03 (m, 2H), 0.33−0.40 (m, 2H), 0.80−0.90 (m, 1H), 1.21 (t, J = 7.2 Hz, 3H), 1.60−1.70 (m, 1H), 1.88−1.98 (m, 1H), 1.99 (s, 3H), 2.23−2.32 (m, 1H), 2.40 (s, 3H), 2.76 (q, J = 7.2 Hz, 2H), 2.92 (d, J = 6.4 Hz, 2H), 3.06−3.13 (m, 1H), 3.21−3.28 (m, 1H), 3.60−3.65 (m, 1H), 3.66−3.72 (m, 4H), 3.73−3.86 (m, 2H), 6.52 (br d, J = 6.8 Hz, 1H), 6.70 (s, 1H), 6.78 (s, 1H), 7.03 (dd, J = 6.8, 8.8 Hz, 1H), 7.45 (br d, J = 8.8 Hz, 1H). 13C NMR (150 MHz, CDCl3) δ 157.6, 154.0, 139.9, 139.0, 137.6, 136.8, 123.4, 121.3, 120.2, 119.6, 115.1, 112.6, 109.9, 67.7, 58.7, 55.9, 38.7, 30.4, 21.8, 19.7, 19.3, 14.4, 10.6, 3.6, 3.6. HRMS calcd for (C27H35N3O2) [M + H]+ 434.2802; found 434.2807. N-(Cyclopropylmethyl)-2-ethyl-7-(2-methoxy-4,6-dimethylphenyl)-N-[(tetrahydrofuran-2-yl)methyl]pyrazolo[1,5-a]pyridin-3-amine (24k). Compound 24k was prepared according to the procedure described for the synthesis of 24h using 23d (40 mg, 0.11 mmol), (2-methoxy-4,6-dimethyllphenyl)boronic acid (38 mg, 0.21 mmol), K2CO3 (44 mg, 0.32 mmol) and Pd(PPh3)4 (19 mg, 0.016 mmol) in DME (1 mL) and water (0.5 mL). The product was purified by column chromatography on silica gel (n-heptane:EtOAc = 11:1) to afford 24k (28 mg, 61%) as a pale yellow oil. 1H NMR (600 MHz, CDCl3) δ −0.06−0.03 (m, 2H), 0.28−0.38 (m, 2H), 0.80−0.91 (m, 1H), 1.17−1.25 (m, 3H), 1.62−1.71 (m, 1H), 1.75−1.95 (m, 3H), 1.97 (br s, 3H), 2.39 (s, 3H), 2.70−2.83 (m, 2H), 2.93−3.00 (m, 2H), 3.03−3.11 (m, 1H), 3.40−3.46 (m, 1H), 3.65−3.74 (m, 1H), 3.67 (s, 3H), 3.80−3.93 (m, 2H), 6.50 (d, J = 6.8 Hz, 1H), 6.69 (s, 1H), 6.76 (s, 1H), 7.01 (dd, J = 6.8, 8.5 Hz, 1H), 7.48 (bd, J = 8.5 Hz, 1H). HRMS calcd for (C27H35N3O2) [M + H]+ 434.2802; found 434.2802. N-(Cyclopropylmethyl)-2-ethyl-7-(2-methoxy-4,6-dimethylphenyl)-N-[(tetrahydro-2H-pyran-4-yl)methyl]pyrazolo[1,5-a]pyridin-3-amine (24l). To a solution of 34a (300 mg, 0.76 mmol) in DMF (5 mL) was added NaH (60%, 46 mg, 1.14 mmol), followed by (bromomethyl)cyclopropane (87 mg, 0.91 mmol) at room temperature, and the mixture was stirred for 1 h at 40 °C. Water was added to the reaction mixture, and the residue was extracted with EtOAc. The organic layer was washed with brine, dried over MgSO4, filtered, and concentrated in vacuo to afford a crude product, which was used in the next step without purification. The crude product from the previous step was dissolved in EtOAc (10 mL). To this mixture was added a 4 M HCl/EtOAc solution (20 mL) at room temperature, and the mixture was stirred for 1 h at 40 °C. A 5 M NaOH aqueous solution was added to the reaction mixture, and the residue was extracted with EtOAc. The organic layer was washed with brine, dried over MgSO4, filtered, and concentrated in vacuo to give crude product, which was used in the next step without purification. The crude product from the previous step was dissolved in THF (2 mL). To this mixture was added tetrahydro-2H-4-pyrancarbaldehyde (173 mg, 1.52 mmol), followed by NaBH(OAc)3 (241 mg, 1.14 mmol), and the mixture was stirred at room temperature for 1 h. A saturated NaHCO3 aqueous solution was added to the reaction mixture, and the residue was extracted with EtOAc. The organic layer was washed with brine, dried over MgSO4, filtered, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (n-hexane:EtOAc = 5:1) to afford 24l (134 mg, 39%, 3 steps) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ −0.04−0.00 (m, 2H), 0.31−0.35 (m, 2H), 0.76−0.88 (m, 1H), 1.20 (t, J = 7.6 Hz, 3H), 1.24−1.34 (m, 2H), 1.54−1.65 (m, 1H), 1.72−1.80 (m, 2H), 1.98 (s, 3H), 2.39 (s, 3H), 2.74 (dq, J = 1.6, 7.6 Hz, 2H), 2.88 (d, J = 6.8 Hz, 5263

dx.doi.org/10.1021/jm300259r | J. Med. Chem. 2012, 55, 5255−5269

Journal of Medicinal Chemistry

Article

N-(Cyclopropylmethyl)-2-ethyl-N-[(tetrahydro-2H-pyran-4yl)methyl]-7-(2,4,5-trimethoxyphenyl)pyrazolo[1,5-a]pyridin3-amine (24p). Compound 24p was prepared according to the procedure described for the synthesis of 24r using 23a (60 mg, 0.15 mmol), (2,4,5-trimethoxyphenyl)boronic acid (39 mg, 0.18 mmol), Pd(PPh3)4 (35 mg, 0.031 mmol) and Ba(OH)2·8H2O (58 mg, 0.18 mmol) in DME (10 mL) and water (5 mL). The product was purified by column chromatography on silica gel (n-hexane:EtOAc = 1:1) to afford 24p (62 mg, 85%) as a yellow oil. 1H NMR (600 MHz, CDCl3) δ −0.02−0.06 (m, 2H), 0.34−0.41 (m, 2H), 0.78−0.88 (m, 1H), 1.19−1.36 (m, 2H), 1.27 (t, J = 7.6 Hz, 3H), 1.52−1.63 (m, 1H), 1.71−1.79 (m, 2H), 2.79 (q, J = 7.6 Hz, 2H), 2.86−2.91 (m, 2H), 3.02−3.07 (m, 2H), 3.27−3.34 (m, 2H), 3.77 (s, 3H), 3.86 (s, 3H), 3.90−3.96 (m, 2H), 3.96 (s, 3H), 6.67 (s, 1H), 6.68 (d, J = 7.0 Hz, 1H), 7.01 (dd, J = 7.0, 8.6 Hz, 1H), 7.22 (s, 1 H), 7.44 (d, J = 8.6 Hz, 1H). 13C NMR (150 MHz, CDCl3) δ 153.5, 152.3, 150.6, 142.9, 137.7, 137.2, 121.3, 120.8, 115.3, 114.5, 114.3, 112.4, 98.3, 68.0, 62.1, 61.9, 56.8, 56.4, 56.1, 34.4, 31.6, 19.8, 14.1, 10.6, 3.7. HRMS calcd for (C28H37N3O4) [M + H]+ 480.2858; found 480.2857. [4-(3-{(Cyclopropylmethyl)[(tetrahydro-2H-pyran-4-yl)methyl]amino}-2-ethylpyrazolo[1,5-a]pyridin-7-yl)-3,5dimethoxyphenyl]methanol (24q). Compound 24q was prepared according to the procedure described for the synthesis of 24r using 23a (250 mg, 0.64 mmol), [4-(hydroxymethyl)-2,6-dimethoxyphenyl]boronic acid (407 mg, 1.9 mmol), Pd(PPh3)4 (111 mg, 0.096 mmol), and Ba(OH)2·8H2O (303 mg, 0.96 mmol) in DME (13 mL) and water (6.5 mL). The product was purified by column chromatography on silica gel (n-hexane:EtOAc = 1:3) to afford 24q (300 mg, 98%) as a pale yellow solid. 1H NMR (400 MHz, CDCl3) δ −0.03−0.05 (m, 2H), 0.32−0.40 (m, 2H), 0.80−0.90 (m, 1H), 1.22 (t, J = 7.5 Hz, 3H), 1.22−1.35 (m, 2H), 1.53−1.66 (m, 1H), 1.72−1.81 (m, 2H), 1.96 (t, J = 5.6 Hz, 1H), 2.78 (q, J = 7.5 Hz, 2H), 2.86−2.92 (m, 2H), 3.02− 3.09 (m, 2H), 3.28−3.38 (m, 2H), 3.74 (s, 6H), 3.90−4.00 (m, 2H), 4.76 (d, J = 5.6 Hz, 2H), 6.61 (dd, J = 1.3, 6.8 Hz, 1H), 6.72 (s, 2H), 7.02 (dd, J = 6.8, 8.8 Hz, 1H), 7.46 (dd, J = 1.3, 8.8 Hz, 1H). 13C NMR (150 MHz, CDCl3) δ 158.8, 153.4, 144.4, 137.4, 134.0, 121.2, 120.6, 115.4, 113.3, 110.9, 102.7, 68.0, 65.2, 62.1, 62.0, 56.1, 34.5, 31.6, 19.6, 14.4, 10.6, 3.6. HRMS calcd for (C28H37N3O4) [M + H]+ 480.2857; found 480.2848. N-(Cyclopropylmethyl)-7-[2,6-dimethoxy-4(methoxymethyl)phenyl]-2-ethyl-N-[(tetrahydro-2H-pyran-4yl)methyl]pyrazolo[1,5-a]pyridin-3-amine (24r). To a solution of 23a (60 mg, 0.15 mmol) in DME (2 mL) and water (1 mL) were added [2,6-dimethoxy-4-(methoxymethyl)phenyl]boronic acid (45 mg, 0.20 mmol), Pd(PPh3)4 (35 mg, 0.030 mmol), and Ba(OH)2·8H2O (72 mg, 0.23 mmol). The mixture was stirred at 90 °C for 4 h under a nitrogen stream and was then cooled and diluted with water. It was subsequently filtered through a pad of Celite to remove insoluble residue, and the filtrate was washed with EtOAc. The mixture was extracted with EtOAc, and the organic layer was washed with brine, dried over MgSO4, filtered, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (nhexane:EtOAc = 1:1) to afford 24r (40 mg, 54%) as a light-yellow solid. 1H NMR (400 MHz, CDCl3) δ −0.02−0.04 (m, 2H), 0.34−0.38 (m, 2H), 0.80−0.90 (m, 1H), 1.22 (t, J = 7.6 Hz, 3H), 1.24−1.34 (m, 2H), 1.54−1.64 (m, 1H), 1.74−1.80 (m, 2H), 2.77 (q, J = 7.6 Hz, 2H), 2.88 (d, J = 6.8 Hz, 2H), 3.05 (d, J = 7.2 Hz, 2H), 3.31 (t, J = 11.6 Hz, 2H), 3.49 (s, 3H), 3.73 (s, 6H), 3.90−4.00 (m, 2H), 4.53 (s, 2H), 6.59 (dd, J = 1.2, 6.8 Hz, 1H), 6.67 (s, 2H), 7.00 (dd, J = 6.8, 8.8 Hz, 1H), 7.44 (dd, J = 1.2, 8.8 Hz, 1H). 13C NMR (150 MHz, CDCl3) δ 158.7, 153.3, 141.4, 137.3, 134.0, 121.0, 120.4, 115.4, 113.1, 111.3, 103.6, 74.9, 68.0, 62.2, 62.0, 58.4, 56.1, 34.5, 31.6, 19.7, 14.3, 10.6, 3.6. HRMS calcd for (C29H39N3O4) [M + H]+ 494.3013; found 494.3019. N-(Cyclopropylmethyl)-7-[2,4-dimethoxy-6(methoxymethyl)phenyl]-2-ethyl-N-[(tetrahydro-2H-pyran-4yl)methyl]pyrazolo[1,5-a]pyridin-3-amine (24s). Compound 24s was prepared according to the procedure described for the synthesis of 24r using 23a (60 mg, 0.15 mmol), [2,4-dimethoxy-6(methoxymethyl)phenyl]boronic acid (104 mg, 0.46 mmol), Pd(PPh3)4 (35 mg, 0.030 mmol) and Ba(OH)2·8H2O (72 mg, 0.23 mmol) in DME (10 mL) and water (5 mL). The product was purified

2H), 3.04 (d, J = 6.8 Hz, 2H), 3.31 (dt, J = 2.0, 11.6 Hz, 2H), 3.68 (s, 3H), 3.92−3.98 (m, 2H), 6.51 (dd, J = 1.6, 6.8 Hz, 1H), 6.69 (s, 1H), 6.77 (s, 1H), 7.01 (dd, J = 6.8, 8.8 Hz, 1H), 7.44 (dd, J = 1.6, 8.8 Hz, 1H). 13C NMR (150 MHz, CDCl3) δ 157.6, 153.9, 139.9, 139.0, 137.5, 136.7, 123.4, 121.2, 120.6, 120.3, 115.2, 112.6, 109.9, 68.0, 62.0, 55.9, 34.5, 31.6, 21.8, 19.7, 19.3, 14.4, 10.6, 3.6. HRMS calcd for (C28H37N3O2) [M + H]+ 448.2959; found 448.2951. N-(Cyclopropylmethyl)-7-(2,6-dimethoxy-4-methylphenyl)2-ethyl-N-[(tetrahydro-2H-pyran-4-yl)methyl]pyrazolo[1,5-a]pyridin-3-amine (24m). Compound 24m was prepared according to the procedure described for the synthesis of 24l using 34b (420 mg, 1.0 mmol), NaH (60%, 61 mg, 1.5 mmol), (bromomethyl)cyclopropane (117 μL, 1.2 mmol) in DMF (5 mL) and 4 M HCl/ EtOAc (40 mL) in EtOAc (20 mL) and tetrahydro-2H-4pyrancarbaldehyde (232 mg, 2.0 mmol), NaBH(OAc)3 (432 mg, 2.0 mmol) in THF (5 mL). The product was purified by column chromatography on silica gel (n-hexane:EtOAc = 4:1) to afford 24m (353 mg, 76%, 3 steps) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ −0.01−0.03 (m, 2H), 0.34−0.40 (m, 2H), 0.80−0.90 (m, 1H), 1.23 (t, J = 7.6 Hz, 3H), 1.20−1.34 (m, 2H), 1.56−1.62 (m, 1H), 1.74−1.80 (m, 2H), 2.45 (s, 3H), 2.77 (q, J = 7.6 Hz, 2H), 2.89 (d, J = 6.4 Hz, 2H), 3.06 (d, J = 6.8 Hz, 2H), 3.32 (dt, J = 2.0, 11.6 Hz, 2H), 3.71 (s, 6H), 3.92−3.98 (m, 2H), 6.53 (s, 2H), 6.60 (dd, J = 1.2, 6.8 Hz, 1H), 7.01 (dd, J = 6.8, 8.8 Hz, 1H), 7.45 (dd, J = 1.2, 8.8 Hz, 1H). 13C NMR (150 MHz, C5D5N) δ 158.4, 152.8, 140.9, 137.2, 134.8, 121.1, 120.8, 115.2, 113.2, 109.5, 105.0, 67.2, 61.5, 61.3, 55.1, 34.1, 31.3, 21.4, 19.5, 13.8, 10.4, 3.4. HRMS calcd for (C28H37N3O3) [M + H]+ 464.2908; found 464.2908. N-(Cyclopropylmethyl)-7-(2,4-dimethoxy-6-methylphenyl)2-ethyl-N-[(tetrahydro-2H-pyran-4-yl)methyl]pyrazolo[1,5-a]pyridin-3-amine (24n). Compound 24n was prepared according to the procedure described for the synthesis of 24l using 34c (80 mg, 0.19 mmol), NaH (60%, 12 mg, 0.29 mmol), (bromomethyl)cyclopropane (22 mg, 0.23 mmol) in DMF (2 mL) and 4 M HCl/ EtOAc (2 mL) in EtOAc (1 mL) and tetrahydro-2H-4-pyrancarbaldehyde (66 mg, 0.58 mmol), NaBH(OAc)3 (123 mg, 0.58 mmol) in THF (2 mL). The product was purified by column chromatography on silica gel (n-hexane:EtOAc = 4:1) to afford 24n (70 mg, 78%, 3 steps) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ −0.05−0.00 (m, 2H), 0.31−0.36 (m, 2H), 0.77−0.87 (m, 1H), 1.21 (t, J = 7.6 Hz, 3H), 1.22−1.32 (m, 2H), 1.56−1.64 (m, 1H), 1.72−1.80 (m, 2H), 2.00 (s, 3H), 2.72−2.79 (m, 2H), 2.87 (d, J = 6.4 Hz, 2H), 3.04 (d, J = 6.8 Hz, 2H), 3.27−3.35 (m, 2H), 3.67 (s, 3H), 3.85 (s, 3H), 3.90−3.96 (m, 2H), 6.43 (d, J = 2.0 Hz, 1H), 6.47 (d, J = 2.0 Hz, 1H), 6.49 (dd, J = 1.2, 6.8 Hz, 1H), 6.99 (dd, J = 6.8, 8.8 Hz, 1H), 7.43 (dd, J = 1.2, 8.8 Hz, 1H). 13C NMR (150 MHz, CDCl3) δ 161.0, 158.9, 153.8, 140.2, 137.5, 136.6, 121.2, 120.6, 115.9, 115.2, 112.9, 106.8, 96.7, 68.0, 62.0, 55.9, 55.3, 34.5, 31.6, 31.6, 19.8, 19.7, 14.4, 10.6, 3.6. HRMS calcd for (C28H37N3O3) [M + H]+ 464.2908; found 464.2913. N-(Cyclopropylmethyl)-2-ethyl-N-[(tetrahydro-2H-pyran-4yl)methyl]-7-(2,4,6-trimethoxyphenyl)pyrazolo[1,5-a]pyridin3-amine (24o). Compound 24o was prepared according to the procedure described for the synthesis of 24l using 34d (100 mg, 0.23 mmol), NaH (60%, 14 mg, 0.35 mmol), (bromomethyl)cyclopropane (27 μL, 0.28 mmol) in DMF (2 mL) and 4 M HCl/EtOAc (2 mL) in EtOAc (1 mL) and tetrahydro-2H-4-pyrancarbaldehyde (107 mg, 0.94 mmol), NaBH(OAc)3 (198 mg, 0.94 mmol) in THF (2 mL). The product was purified by column chromatography on silica gel (nhexane:EtOAc = 3:1) to afford 24o (82 mg, 73%, 3 steps) as a pale yellow solid. 1H NMR (400 MHz, CDCl3) δ −0.02−0.03 (m, 2H), 0.34−0.40 (m, 2H), 0.80−0.90 (m, 1H), 1.23 (t, J = 7.6 Hz, 3H), 1.23−1.34 (m, 2H), 1.54−1.64 (m, 1H), 1.73−1.80 (m, 2H), 2.78 (q, J = 7.6 Hz, 2H), 2.89 (d, J = 6.8 Hz, 2H), 3.05 (d, J = 6.8 Hz, 2H), 3.27−3.36 (m, 2H), 3.71 (s, 6H), 3.89 (s, 3H), 3.92−3.97 (m, 2H), 6.26 (s, 2H), 6.59 (dd, J = 1.6, 6.8 Hz, 1H), 7.00 (dd, J = 6.8, 8.8 Hz, 1H), 7.43 (dd, J = 1.6, 8.8 Hz, 1H). 13C NMR (150 MHz, CDCl3) δ 162.2, 159.5, 153.2, 137.4, 134.1, 121.0, 120.4, 115.2, 113.4, 105.2, 91.4, 68.0, 62.2, 62.0, 56.0, 55.4, 34.5, 31.6, 19.7, 14.4, 10.6, 3.6. HRMS calcd for (C28H37N3O4) [M + H]+ 480.2857; found 480.2862. 5264

dx.doi.org/10.1021/jm300259r | J. Med. Chem. 2012, 55, 5255−5269

Journal of Medicinal Chemistry

Article

2.97 (dd, J = 7.3, 7.3 Hz, 2H), 3.26−3.35 (m, 2H), 3.70 (s, 6H), 3.90− 3.97 (m, 2H), 6.51 (s, 2H), 6.59 (dd, J = 1.3, 6.8 Hz, 1H), 7.00 (dd, J = 6.8, 8.8 Hz, 1H), 7.41 (dd, J = 1.3, 8.8 Hz, 1H). 13C NMR (150 MHz, CDCl3) δ 158.5, 153.3, 141.1, 137.3, 134.2, 121.0, 120.3, 115.2, 113.2, 109.3, 105.5, 68.0, 62.8, 59.1, 56.0, 34.5, 31.7, 22.4, 22.1, 19.7, 14.5, 11.7. HRMS calcd for (C27H37N3O3) [M + H]+ 452.2908; found 452.2900. 7-[2,6-Dimethoxy-4-(methoxymethyl)phenyl]-2-ethyl-Npropyl-N-[(tetrahydro-2H-pyran-4-yl)methyl]pyrazolo[1,5-a]pyridin-3-amine (24w). Compound 24w was prepared according to the procedure described for the synthesis of 24l using 34e (70 mg, 0.16 mmol), NaH (60%, 10 mg, 0.24 mmol), 1-bromopropane (17 μL, 0.19 mmol) in DMF (2 mL) and 4 M HCl/EtOAc (2 mL) in EtOAc (1 mL) and tetrahydro-2H-4-pyrancarbaldehyde (48 mg, 0.42 mmol), NaBH(OAc)3 (90 mg, 0.42 mmol) in THF (2 mL). The product was purified by column chromatography on silica gel (n-hexane:EtOAc = 2:1) to afford 24w (49 mg, 63%, 3 steps) as a light yellow solid. 1H NMR (400 MHz, CDCl3) δ 0.87 (t, J = 7.6 Hz, 3H), 1.20 (t, J = 7.2 Hz, 3H), 1.23−1.32 (m, 2H), 1.36−1.45 (m, 2H), 1.52−.62 (m, 1H), 1.72−1.78 (m, 2H), 2.73 (q, J = 7.2 Hz, 2H), 2.96−3.00 (m, 4H), 3.27−3.35 (m, 2H), 3.49 (s, 3H), 3.73 (s, 6H), 3.90−3.97 (m, 2H), 4.53 (s, 2H), 6.60 (dd, J = 1.6, 6.8 Hz, 1H), 6.68 (s, 2H), 7.01 (dd, J = 6.8, 8.8 Hz, 1H), 7.42 (dd, J = 1.6, 8.8 Hz, 1H). 13C NMR (150 MHz, CDCl3) δ 158.7, 153.3, 141.4, 137.3, 134.0, 121.0, 120.3, 115.3, 113.2, 111.2, 103.6, 74.9, 68.0, 62.8, 59.1, 58.5, 56.1, 34.5, 31.7, 22.1, 19.7, 14.4, 11.7. HRMS calcd for (C28H39N3O4) [M + H]+ 482.3013; found 482.3005. N-(Cyclopropylmethyl)-7-[2,6-dimethoxy-4(methoxymethyl)phenyl]-2-methoxy-N-[(tetrahydro-2Hpyran-4-yl)methyl]pyrazolo[1,5-a]pyridin-3-amine (27a). Compound 27a was prepared according to the procedure described for the synthesis of 24r using 26 (48 mg, 0.12 mmol), [2,6-dimethoxy-4(methoxymethyl)phenyl]boronic acid (36 mg, 0.16 mmol), Pd(PPh3)4 (28 mg, 0.024 mmol) and Ba(OH)2·8H2O (58 mg, 0.18 mmol) in DME (2 mL) and water (1 mL). The product was purified by column chromatography on silica gel (n-hexane:EtOAc = 2:1), follwed by column chromatography on NH silica gel (n-hexane:EtOAc = 5:1) to afford 27a (53 mg, 89%) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ −0.02−0.04 (m, 2H), 0.30−0.36 (m, 2H), 0.80−0.92 (m, 1H), 1.24−1.36 (m, 2H), 1.52−1.64 (m, 1H), 1.74−1.82 (m, 2H), 2.84 (d, J = 6.8 Hz, 2H), 2.97 (d, J = 6.8 Hz, 2H), 3.32 (td, J = 2.0, 11.6 Hz, 2H), 3.51 (s, 3H), 3.76 (s, 6H), 3.87 (s, 3H), 3.90−3.98 (m, 2H), 4.55 (s, 2H), 6.51 (dd, J = 1.6, 6.8 Hz, 1H), 6.69 (s, 2H), 7.04 (dd, J = 7.2, 8.8 Hz, 1H), 7.33 (dd, J = 1.6, 8.8 Hz, 1H). 13C NMR (150 MHz, CDCl3) δ 161.2, 158.8, 141.3, 139.2, 133.7, 122.2, 114.0, 111.5, 111.3, 105.9, 103.4, 75.0, 68.1, 61.7, 61.2, 58.5, 56.1, 56.0, 34.2, 31.6, 10.4, 3.4. HRMS calcd for (C28H37N3O5) [M + H]+ 496.2806; found 496.2798. N-(Cyclopropylmethyl)-7-(2,6-dimethoxy-4-methylphenyl)2-methoxy-N-[(tetrahydro-2H-pyran-4-yl)methyl]pyrazolo[1,5a]pyridin-3-amine (27b). Compound 27b was prepared according to the procedure described for the synthesis of 24l using 36a (250 mg, 0.61 mmol), NaH (60%, 36 mg, 0.91 mmol), (bromomethyl)cyclopropane (90 μL, 0.91 mmol) in DMF (8 mL) and 4 M HCl/ EtOAc (15 mL) in EtOAc (10 mL) and tetrahydro-2H-4pyrancarbaldehyde (72 mg, 0.63 mmol), NaBH(OAc)3 (24 mg, 0.63 mmol) in THF (5 mL). The product was purified by column chromatography on silica gel (n-hexane:EtOAc = 4:1) to afford 27b (45 mg, 16%, 3 steps) as a yellow solid. 1H NMR (400 MHz, CDCl3) δ −0.04−0.08 (m, 2H), 0.28−0.40 (m, 2H), 0.80−0.94 (m, 1H), 1.20−1.38 (m, 2H), 1.52−1.70 (m, 1H), 1.74−1.84 (m, 2H), 2.47 (s, 3H), 2.84 (d, J = 6.6 Hz, 2H), 2.97 (d, J = 6.9 Hz, 2H), 3.33 (dt, J = 2.0, 12.0 Hz, 2H), 3.75 (s, 6H), 3.89 (s, 3H), 3.90−4.00 (m, 2H), 6.52 (dd, J = 1.4, 6.9 Hz, 1H), 6.54 (s, 2H), 7.04 (dd, J = 6.8, 8.8 Hz, 1H), 7.33 (dd, J = 1.4, 8.8 Hz, 1H). 13C NMR (150 MHz, CDCl3) δ 161.2, 158.6, 141.0, 139.2, 133.9, 122.2, 113.8, 111.6, 109.4, 105.8, 105.3, 68.1, 61.7, 61.2, 56.0, 56.0, 34.2, 31.6, 22.4, 10.4, 3.4. HRMS calcd for (C27H35N3O4) [M + H]+ 466.2700; found 466.2694. N-(Cyclopropylmethyl)-7-iodo-2-(methylthio)pyrazolo[1,5a]pyridin-3-amine (28). Compound 28 was prepared according to

by column chromatography on silica gel (n-hexane:EtOAc = 3:1), follwed by column chromatography on NH silica gel (n-hexane:EtOAc = 3:1) to afford 24s (38 mg, 50%) as a pale yellow solid. 1H NMR (400 MHz, CDCl3) δ −0.04−0.02 (m, 2H), 0.30−0.36 (m, 2H), 0.78−0.88 (m, 1H), 1.20 (t, J = 7.6 Hz, 3H), 1.20−1.32 (m, 2H), 1.52−1.64 (m, 1H), 1.72−1.80 (m, 2H), 2.70−2.80 (m, 2H), 2.87 (d, J = 6.8 Hz, 2H), 3.04 (d, J = 6.8 Hz, 2H), 3.19 (s, 3H), 3.28−3.34 (m, 2H), 3.68 (s, 3H), 3.88 (s, 3H), 3.90−3.98 (m, 2H), 3.98 (d, J = 12.8 Hz, 1H), 4.21 (d, J = 12.8 Hz, 1H), 6.51 (d, J = 2.4 Hz, 1H), 6.54 (dd, J = 1.6, 6.8 Hz, 1H), 6.76 (d, J = 2.4 Hz, 1H), 6.99 (dd, J = 6.8, 8.8 Hz, 1H), 7.44 (dd, J = 1.2, 8.8 Hz, 1H). 13C NMR (150 MHz, CDCl3) δ 161.5, 158.7, 153.8, 140.4, 137.5, 135.4, 121.1, 120.7, 115.5, 114.7, 113.1, 103.8, 98.5, 71.9, 68.0, 62.0, 62.0, 58.1, 56.0, 55.4, 34.5, 31.6, 31.6, 19.7, 14.3, 10.6, 3.6, 3.6. HRMS calcd for (C29H39N3O4) [M + H]+ 494.3013; found 494.3019. N-(Cyclopropylmethyl)-7-[4-(ethoxymethyl)-2,6-dimethoxyphenyl]-2-ethyl-N-[(tetrahydro-2H-pyran-4-yl)methyl]pyrazolo[1,5-a]pyridin-3-amine (24t). Compound 24t was prepared according to the procedure described for the synthesis of 24r using 23a (67 mg, 0.17 mmol), [4-(ethoxymethyl)-2,6dimethoxyphenyl]boronic acid (104 mg, 0.46 mmol), Pd(PPh3)4 (40 mg, 0.034 mmol) and Ba(OH)2·8H2O (65 mg, 0.21 mmol) in DME (10 mL) and water (5 mL). The product was purified by column chromatography on silica gel (n-hexane:EtOAc = 2:1), follwed by column chromatography on NH silica gel (n-hexane:EtOAc = 4:1) to afford 24t (61 mg, 70%) as a yellow solid. 1H NMR (400 MHz, CDCl3) δ −0.02−0.06 (m, 2H), 0.34−0.43 (m, 2H), 0.80−0.94 (m, 1H), 1.24 (t, J = 7.5 Hz, 3H), 1.33 (t, J = 7.0 Hz, 3H), 1.20−1.38 (m, 2H), 1.54−1.68 (m, 1H), 1.74−1.84 (m, 2H), 2.78 (q, J = 7.5 Hz, 2H), 2.90 (d, J = 6.6 Hz, 2H), 3.07 (d, J = 7.0 Hz, 2H), 3.33 (dt, J = 1.6, 12.0 Hz, 2H), 3.66 (q, J = 7.0 Hz, 2H), 3.75 (s, 6H), 3.92−4.02 (m, 2H), 4.59 (s, 2H), 6.61 (br d, J = 6.8 Hz, 1H), 6.71 (s, 2H), 7.03 (dd, J = 7.0, 8.8 Hz, 1H), 7.46 (br d, J = 8.8 Hz, 1H). 13C NMR (150 MHz, CDCl3) δ 158.7, 153.3, 141.8, 137.3, 134.0, 121.0, 120.4, 115.4, 113.1, 111.2, 103.7, 73.0, 68.0, 66.0, 62.2, 62.0, 56.1, 34.5, 31.6, 19.7, 15.3, 14.3, 10.6, 3.6. HRMS calcd for (C30H41N3O4) [M + H]+ 508.3170; found 508.3167. N-(Cyclopropylmethyl)-7-(2,6-dimethoxyphenyl)-2-ethyl-N[(tetrahydro-2H-pyran-4-yl)methyl]pyrazolo[1,5-a]pyridin-3amine (24u). Compound 24u was prepared according to the procedure described for the synthesis of 24r using 23a (50 mg, 0.12 mmol), (2,6-dimethoxyphenyl)boronic acid (33 mg, 0.18 mmol), Pd(PPh3)4 (21 mg, 0.018 mmol) and Ba(OH)2·8H2O (57 mg, 0.18 mmol) in DME (2.4 mL) and water (1.2 mL). The product was purified by column chromatography on NH silica gel (nhexane:EtOAc = 5:1), follwed by column chromatography on silica gel (n-hexane:EtOAc = 2:1) to afford 24u (46 mg, 85%) as a pale yellow oil. 1H NMR (400 MHz, CDCl3) δ −0.05−0.05 (m, 2H), 0.31−0.40 (m, 2H), 0.79−0.90 (m, 1H), 1.20−1.36 (m, 2H), 1.21 (t, J = 7.6 Hz, 3H), 1.53−1.65 (m, 1H), 1.71−1.80 (m, 2H), 2.76 (q, J = 7.6 Hz, 2H), 2.85−2.91 (m, 2H), 3.01−3.08 (m, 2H), 3.26−3.35 (m, 2H), 3.71 (s, 6H), 3.87−4.00 (m, 2H), 6.60 (d, J = 6.4 Hz, 1H), 6.69 (d, J = 8.4 Hz, 2H), 7.01 (dd, J = 6.4, 8.7 Hz, 1H), 7.39 (t, J = 8.4 Hz, 1H), 7.44 (d, J = 8.7 Hz, 1H). 13C NMR (150 MHz, CDCl3) δ 158.8, 153.3, 137.3, 134.1, 130.7, 121.0, 120.5, 115.4, 113.1, 112.2, 104.6, 68.0, 62.2, 62.0, 56.1, 34.5, 31.6, 19.7, 14.3, 10.6, 3.6. HRMS calcd for (C27H35N3O3) [M + H]+ 450.2751; found 450.2754. 7-(2,6-Dimethoxy-4-methylphenyl)-2-ethyl-N-propyl-N-[(tetrahydro-2H-pyran-4-yl)methyl]pyrazolo[1,5-a]pyridin-3-amine (24v). Compound 24v was prepared according to the procedure described for the synthesis of 24l using 34b (120 mg, 0.29 mmol), NaH (60%, 16 mg, 0.40 mmol), iodopropane (34 μL, 0.35 mmol) in DMF (0.8 mL) and 4 M HCl/EtOAc (0.8 mL) and tetrahydro-2H-4pyrancarbaldehyde (78 mg, 0.68 mmol), NaBH(OAc)3 (87 mg, 0.41 mmol) in THF (1.4 mL). The residue was purified by column chromatography on silica gel (n-hexane:EtOAc = 5:2) to afford 24v (111 mg, 86%, 3 steps) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ 0.87 (t, J = 7.3 Hz, 3H), 1.20 (t, J = 7.6 Hz, 3H), 1.22−1.32 (m, 2H), 1.39 (ddq, J = 7.3, 7.3, 7.3 Hz, 2H), 1.51−1.63 (m, 1H), 1.70−1.78 (m, 2H), 2.43 (s, 3H), 2.74 (q, J = 7.6 Hz, 2H), 2.93−2.98 (m, 2H), 5265

dx.doi.org/10.1021/jm300259r | J. Med. Chem. 2012, 55, 5255−5269

Journal of Medicinal Chemistry

Article

the procedure described for the synthesis of 22a using 21 (600 mg, 1.5 mmol), NaH (60%, 80 mg, 2.0 mmol), (bromomethyl)cyclopropane (0.22 mL, 2.2 mmol) in DMF (6 mL) and 4 M HCl/EtOAc (10 mL, 40 mmol) in EtOAc (1 mL). The product was purified by column chromatography on silica gel (n-hexane:EtOAc = 3:1) to afford 28 (506 mg, 94%, 2 steps) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ 0.16−0.24 (m, 2H), 0.48−0.56 (m, 2H), 1.00−1.10 (m, 1H), 2.60 (s, 3H), 2.96 (d, J = 6.0 Hz, 2H), 3.00−3.24 (m, 1H), 6.68 (ddd, J = 1.2, 6.8, 8.8 Hz, 1H), 7.17 (dd, J = 1.2, 6.8 Hz, 1H), 7.43 (dd, J = 1.2, 8.8 Hz, 1H). N-(Cyclopropylmethyl)-7-iodo-2-(methylthio)-N-[(tetrahydro-2H-pyran-4-yl)methyl]pyrazolo[1,5-a]pyridin-3-amine (29). Compound 29 (50 mg, 58%) was obtained as a yellow oil according to the procedure described for the synthesis of 23a using 28 (300 mg, 1.0 mmol), tetrahydro-2H-pyran-4-carbaldehyde (56 mg, 0.49 mmol) and NaBH(OAc)3 (103 mg, 0.49 mmol) in THF (2.5 mL), which was used in the next step without purification. 1H NMR (400 MHz, CDCl3) δ −0.06−0.04 (m, 2H), 0.30−0.38 (m, 2H), 0.74−0.86 (m, 1H), 1.20−1.32 (m, 2H), 1.40−1.60 (m, 1H), 1.66− 1.80 (m, 2H), 2.69 (s, 3H), 2.85 (d, J = 6.8 Hz, 2H), 3.02 (d, J = 7.2 Hz, 2H), 3.22−3.32 (m, 2H), 3.86−3.94 (m, 2H), 6.72 (dd, J = 7.2, 8.8 Hz, 1H), 7.15 (dd, J = 1.2, 7.2 Hz, 1H), 7.40 (dd, J = 1.2, 8.8 Hz, 1H). 13C NMR (150 MHz, CDCl3) δ 148.3, 139.0, 123.0, 122.5, 121.8, 115.6, 92.4, 67.9, 61.1, 60.7, 34.2, 31.5, 14.3, 10.2, 3.6. HRMS calcd for (C18H24IN3OS) [M + H]+ 458.0758; found 458.0764. N-(Cyclopropylmethyl)-7-[2,6-dimethoxy-4(methoxymethyl)phenyl]-2-(methylthio)-N-[(tetrahydro-2Hpyran-4-yl)methyl]pyrazolo[1,5-a]pyridin-3-amine (30a). Compound 30a was prepared according to the procedure described for the synthesis of 24r using 29 (50 mg, 0.19 mmol), [2,6-dimethoxy-4(methoxymethyl)phenyl]boronic acid (50 mg, 0.25 mmol), Pd(PPh3)4 (40 mg, 0.038 mmol) and Ba(OH)2·8H2O (56 mg, 0.18 mmol) in DME (2 mL) and water (1 mL). The product was purified by column chromatography on silica gel (n-hexane:EtOAc = 3:1), follwed by column chromatography on NH silica gel (n-hexane:EtOAc = 6:1) to afford 30a (36 mg, 37%) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ −0.02−0.04 (m, 2H), 0.30−0.38 (m, 2H), 0.82−0.92 (m, 1H), 1.22−1.34 (m, 2H), 1.52−1.64 (m, 1H), 1.76−1.82 (m, 2H), 2.44 (s, 3H), 2.90 (d, J = 6.8 Hz, 2H), 3.06 (d, J = 7.2 Hz, 2H), 3.32 (td, J = 2.0, 12.0 Hz, 2H), 3.50 (s, 3H), 3.74 (s, 6H), 3.90−3.98 (m, 2H), 4.54 (s, 2H), 6.59 (dd, J = 1.6, 7.2 Hz, 1H), 6.67 (s, 2H), 7.05 (dd, J = 7.2, 8.8 Hz, 1H), 7.41 (dd, J = 1.6, 8.8 Hz, 1H). 13C NMR (150 MHz, CDCl3) δ 158.8, 146.3, 141.5, 138.7, 133.8, 121.9, 120.8, 114.6, 113.0, 110.9, 103.2, 74.9, 68.0, 61.3, 61.2, 58.4, 56.0, 34.3, 31.6, 14.8, 10.4, 3.5. HRMS calcd for (C28H37N3O4S) [M + H]+ 512.2578; found 512.2582. N-(cyclopropylmethyl)-7-[2,6-dimethoxy-4(methoxymethyl)phenyl]-2-methyl-N-[(tetrahydro-2H-pyran4-yl)methyl]pyrazolo[1,5-a]pyridin-3-amine (33). Compound 33 was prepared according to the procedure described for the synthesis of 24h using 32 (40 mg, 0.11 mmol), [2,6-dimethoxy-4(methoxymethyl)phenyl]boronic acid (48 mg, 0.21 mmol), K2CO3 (44 mg, 0.32 mmol), and Pd(PPh3)4 (18 mg, 0.016 mmol) in DME (1 mL) and water (0.5 mL). The product was purified by column chromatography on silica gel (n-heptane:EtOAc = 45:55), follwed by column chromatography on NH silica gel (n-heptane:EtOAc = 7:3) to afford 33 (31 mg, 61%) as a pale yellow solid. 1H NMR (400 MHz, CDCl3) δ −0.05−0.02 (m, 2H), 0.31−0.36 (m, 2H), 0.78−0.88 (m, 1H), 1.20−1.32 (m, 2H), 1.54−1.64 (m, 1H), 1.72−1.78 (m, 2H), 2.34 (s, 3H), 2.85 (d, J = 7.2 Hz, 2H), 3.03 (d, J = 7.2 Hz, 2H), 3.26− 3.34 (m, 2H), 3.47 (s, 3H), 3.71 (s, 6H), 3.90−3.96 (m, 2H), 4.51 (s, 2H), 6.53 (dd, J = 1.2, 6.8 Hz, 1H), 6.66 (s, 2H), 7.00 (dd, J = 6.8, 8.8 Hz, 1H), 7.42 (dd, J = 1.2, 8.8 Hz, 1H). 13C NMR (150 MHz, CDCl3) δ 158.7, 148.0, 141.5, 137.6, 133.9, 121.3, 121.0, 115.1, 113.0, 111.3, 103.6, 74.9, 68.0, 61.9, 61.7, 58.4, 56.1, 34.4, 31.6, 12.7, 10.5, 3.5. HRMS calcd for (C28H37N3O4) [M + H]+ 480.2857; found 480.2856. tert-Butyl [2-ethyl-7-(2-methoxy-4,6-dimethylphenyl)pyrazolo[1,5-a]pyridin-3-yl]carbamate (34a). Compound 34a was prepared according to the procedure described for the synthesis of 24h using 10 (40 mg, 0.12 mmol), (2-methoxy-4,6-

dimethyllphenyl)boronic acid (34 mg, 0.19 mmol), K2CO3 (26 mg, 0.19 mmol), and Pd(PPh3)4 (20 mg, 0.017 mmol) in DME (1 mL) and water (0.5 mL). The product was purified by column chromatography on silica gel (n-heptane:EtOAc = 4:1) to afford 34a (40 mg, 86%) as a pale yellow solid. 1H NMR (400 MHz, CDCl3) δ 1.21 (t, J = 7.6 Hz, 3 H), 1.54 (br s., 6H), 1.98 (s, 3H), 2.39 (s, 3H), 2.73 (q, J = 7.6 Hz, 2H), 3.65 (s, 3H), 5.88 (br s., 1H), 6.56 (d, J = 6.6 Hz, 1H), 6.68 (s, 1H), 6.76 (s, 1H), 7.13 (dd, J = 8.8, 6.6 Hz, 1H), 7.37 (d, J = 8.8 Hz, 1H). 13C NMR (150 MHz, C5D5N) δ 157.6, 155.0, 151.7, 139.6, 138.5, 136.7, 136.6, 122.6, 122.0, 120.0, 114.8, 112.8, 109.4, 107.8, 78.3, 54.9, 27.8, 20.9, 19.6, 18.7, 13.1. HRMS calcd for (C23H30N3O3) [M + H]+ 396.2282; found 396.2278. 7-(2-Chloro-4-methoxyphenyl)-2-ethyl-3-nitropyrazolo[1,5a]pyridine (40a). Compound 40a was prepared according to the procedure described for the synthesis of 24r using 9 (94 mg, 0.35 mmol), (2-chloro-4-methoxyphenyl)boronic acid (130 mg, 0.70 mmol), Pd(PPh3)4 (80 mg, 0.070 mmol) and Ba(OH)2·8H2O (220 mg, 0.70 mmol) in DME (15 mL) and water (7.5 mL). The product was purified by column chromatography on silica gel (n-hexane:EtOAc = 5:1) to afford 40a (115 mg, quantitative yield) as a yellow solid. 1H NMR (400 MHz, CDCl3) δ 1.30 (t, J = 7.6 Hz, 3H), 3.15 (q, J = 7.6 Hz, 2H), 3.90 (s, 3H), 6.96 (dd, J = 2.4, 8.4 Hz, 1H), 7.07 (dd, J = 1.6, 7.2 Hz, 1H), 7.10 (d, J = 2.4 Hz, 1H), 7.40 (d, J = 8.4 Hz, 1H), 7.68 (dd, J = 7.2, 8.8 Hz, 1H), 8.40 (dd, J = 1.6, 8.8 Hz, 1H). 2-(Methylsulfonyl)-3-nitropyrazolo[1,5-a]pyridine (41). A mixture of aluminum oxide (57.2 g, 561 mmol) and water (11.4 mL) was stirred to mix homogeneously. To the mixture were added 19 (14.3 g, 68.4 mmol) and CHCl3 (343 mL), followed by potassium peroxomonosulfate compound (126 g, 205 mmol) at room temperature, and the reaction mixture was stirred for 8 h at 80 °C. The reaction mixture was cooled and filtered through a pad of Celite to remove insoluble residue The filtrate was concentrated in vacuo to give a crude product of 41 (13.7 g, 83%) as a yellow solid. 1H NMR (400 MHz, CDCl3) δ 4.21 (s, 3H), 7.08 (dd, J = 6.8, 8.4 Hz, 1H), 7.63 (dd, J = 8.4, 8.8 Hz, 1H), 8.27 (d, J = 8.8 Hz, 1H), 8.34 (d, J = 6.8 Hz, 1H). 3-Nitropyrazolo[1,5-a]pyridine-2-carbonitrile (42). To a solution of 41 (2.1 g, 8.6 mmol) in THF (16 mL) and DMF (3 mL) was added sodium cyanide (508 mg, 10.4 mmol) at room temperature, and the reaction mixture was stirred for 6 h at 80 °C. The mixture was cooled and filtered through a pad of Celite to remove insoluble residue. The filtrate was concentrated in vacuo, and then water was added. The resulting solid was collected by filtration and washed with water to give a crude product of 42 (1.1 g, 69%) as a brown solid. 1H NMR (400 MHz, CDCl3) δ 7.34 (dd, J = 6.8, 6.8 Hz, 1H), 7.81 (dd, J = 6.8, 9.2 Hz, 1H), 8.43 (d, J = 9.2 Hz, 1H), 8.61 (d, J = 6.8 Hz, 1H). 3-[(Cyclopropylmethyl)[(tetrahydro-2H-pyran-4-yl)methyl]amino]pyrazolo[1,5-a]pyridine-2-carbonitrile (43). To a solution of 42 (1.1 g, 6.0 mmol) in EtOAc (30 mL) was added 10% Pd/C (0.98 g, 50% wet). The reaction mixture was treated under H2 atmosphere (1 atm) for 2 h at room temperature. The reaction mixture was filtered through a pad of Celite, and the filtrate was concentrated in vacuo to give a crude product (843 mg, 89%) as a yellow solid. The crude product (527 mg, 3.3 mmol) from the previous step was dissolved in THF (11 mL). To this mixture was added cyclopropanecarboxaldehyde (249 μL, 3.3 mmol), followed by titanium(IV) isopropoxide (1.2 mL, 4.0 mmol). After stirring for 7 h at room temperature, to the mixture was added MeOH (11 mL), followed by NaBH4 (126 mg. 3.3 mmol) at 0 °C, and the reaction mixture was stirred for 3 h at room temperature. Water and a small amount of aqueous ammonia solution was added. The mixture was filtered through a pad of Celite to remove insoluble residue, and was extracted with EtOAc. The organic layer was washed with brine, dried over MgSO4, filtered, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (n-heptane:EtOAc = 1:1) to give the secondary amine including impurity. To a solution of impurity-containing product from the previous step in MeOH (7 mL) and AcOH (0.7 mL) were added tetrahydro-2Hpyran-4-carbaldehyde (317 mg, 2.8 mmol) and α-picoline-borane (223 mg, 2.1 mmol) at room temperature, and the reaction mixture was 5266

dx.doi.org/10.1021/jm300259r | J. Med. Chem. 2012, 55, 5255−5269

Journal of Medicinal Chemistry

Article

NMR (100 MHz, C5D5N) δ 159.3, 153.5, 145.8, 142.7, 139.4, 137.9, 135.2, 129.0, 126.9, 121.8, 121.6, 116.1, 113.8, 111.8, 103.7, 74.7, 67.9, 67.9, 62.2, 62.0, 58.2, 55.8, 34.8, 32.0, 32.0, 21.1, 20.2, 14.5, 11.1, 4.1, 4.1. IR (KBr, cm−1) 2629 (w), 1638 (w), 1612 (w), 1583 (w), 1542 (w), 1224 (m), 1159 (m), 1131 (m), 1119 (m), 1031 (w). Anal. Calcd for C36H47N3O7S: C, 64.94%, H, 7.11%, N, 6.31%; Found: C, 64.77%, H, 7.11%, N, 6.22%. Biology. CRF1 Receptor Antagonist Binding Assays. HEK293 cells expressing human CRF1 receptor were cloned using essentially the same method as that described in literature.32 CRF1 receptor binding was performed by using the homogeneous technique of scintillation proximity (SPA, Amersham Pharmacia, UK) with 96-well plates. Cell membrane (5 μg/well), wheat germ agglutinin coated SPA beads (1 mg/well), [125I] human/rat CRF (0.1 nM), and diluted test compound solution were suspended in 150 μL of assay buffer (137 mM NaCl, 8.1 mM Na2HPO4, 2.7 mM KCl, 1.5 mM KH2PO4, 10 mM MgCl2, 2 mM EGTA, 1.5% bovine serum albumin (BSA), pH 7.0). Total binding and nonspecific binding were measured in the absence and presence of 0.2 μM unlabeled sauvagine, respectively. Plates were shaken gently and incubated for more than 2 h at room temperature. After centrifugation (260 g, 5 min, rt), radioactivity was detected using TopCount (Packard, USA, MA) with a 1 min counting time per well. Each count was corrected by subtracting nonspecific binding, and was represented as a percentage of total binding. The IC50 value of each compound was calculated using a concentration−response curve. Compounds 46 and 1 were also examined by filtration methods, as described previously, but with some modification.36 Diluted test compounds and 0.1 nM [125I] human/rat CRF were incubated with cell membrane (5 μg/well) in 150 μL of assay buffer (0.2% BSA, 50 mM Tris-HCl, 10 mM MgCl2, 2 mM EGTA, protease inhibitor cocktail, pH 7.1) for more than 2 h at room temperature. Bound and free radioligand were separated by filtration using a glass fiber filter (Whatman GF/C, presoaked with 1% BSA/PBS(-)). The filter was washed with 5 mL of PBS (-), and was measured with a gammacounter (ARC-1000 M, Aloka, Japan). Nonspecific binding was defined using 0.4 μM sauvagine. The IC50 value of each compound was obtained as described above. The Ki value was calculated with the Cheng−Prusoff equation: Ki = IC50/(1 + L/Kd), where L is the concentration of [125I]-CRF and Kd is the dissociation constant of the radioligand. The Ki value was obtained using MS Excel 2000 (Microsoft Co., Redmond, WA, USA). Functional Assay. To determine the antagonistic activities, their effects on CRF-stimulated intracellular cAMP accumulation was examined in HEK293 cells expressing human CRF1 receptor, which were cloned as described in the literature.32 cAMP was measured using an enzyme immunoassay kit (Amersham Pharmacia, UK) or a cAMP kit, which is a homogeneous time-resolved fluorescence system (CIS bio international, France). Similar results were obtained with these two assay kits. HEK293 cells expressing human CRF1 receptor (50 000 cells/well) in Dulbecco’s modified Eagle medium containing 0.1% fetal bovine serum and 1 mM 3-isobutyl-1-methylxanthine, which is a phosphodiesterase inhibitor, were seeded in 96-well plates. After preincubation for 30 min, diluted test compounds were added and incubated for 30 min at 37 °C. Cells were stimulated with 1 nM human/rat CRF for 30 min at 37 °C and collected by centrifugation (630 g, 5 min, 4 °C). After aspiration of the medium, cells were lysed with lysis buffer, and the amount of intracellular cAMP was measured according to the procedures detailed in the instruction manual of each kit. Each produced cAMP was corrected for basal cAMP production (i.e., in the absence of CRF) and was expressed as a percentage of total production. The IC50 value of each compound was calculated using a concentration−response curve. Light/Dark Test in Mice. Male BALB/c mice (Charles River Japan Inc., Kanagawa, Japan) weighing 20−28 g were used. Test compounds (0.4, 2, 10 mg/kg for 46 and 2, 10, 50 mg/kg for 1) were orally administered to the mice 1 h before the test. A control group received an equivalent volume of vehicle (0.5% methyl cellulose, 10 mL/kg). The test apparatus was a modified version of that described by Belzung et al.37 It consisted of two acrylic boxes, one of which was a black darkened box (dark box; 10 × 15 × 20 cm3 high), and the other

stirred for 16 h. A saturated NaHCO3 aqueous solution was added to the reaction mixture, and the residue was extracted with EtOAc. The organic layer was washed with brine, dried over MgSO4, filtered, and concentrated in vacuo. The residue was purified by column chromatography on NH silica gel (n-heptane:EtOAc = 1:1) to afford 43 (378 mg, 37%, 2 steps) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ −0.03−0.05 (m, 2H), 0.38−0.47 (m, 2H), 0.83−0.95 (m, 1H), 1.22−1.37 (m, 2H), 1.56−1.70 (m, 1H), 1.71−1.81 (m, 2H), 2.96−3.02 (m, 2H), 3.18−3.24 (m, 2H), 3.26−3.35 (m, 2H), 3.90− 3.98 (m, 2H), 6.92 (dd, J = 6.8, 7.2 Hz, 1H), 7.15 (dd, J = 6.8, 9.2 Hz, 1H), 7.58 (d, J = 9.2 Hz, 1H), 8.35 (d, J = 7.2 Hz, 1H). 3-[(Cyclopropylmethyl)[(tetrahydro-2H-pyran-4-yl)methyl]amino]-7-iodopyrazolo[1,5-a]pyridine-2-carbonitrile (44). To a solution of 43 (222 mg, 0.72 mmol) in THF (4 mL) was added n-BuLi (1.59 M hexane solution; 0.54 mL, 0.86 mmol) dropwise at −90 °C under a nitrogen stream, and the reaction mixture was stirred for 30 min at the same temperature. A solution of 1,2-diiodoethane (263 g, 0.93 mmol) in THF (4 mL) was added dropwise to the reaction mixture at −78 °C, and the mixture was allowed to warm to −40 °C over 2 h. The mixture was quenched carefully with a saturated NH4Cl aqueous solution, and the internal temperature was allowed to room temperature. The mixture was extracted with EtOAc, and the organic layer was washed with brine, dried over MgSO4, filtered, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (n-heptane:EtOAc = 2:1) to afford 44 (198 mg, 63%) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ −0.03− 0.06 (m, 2H), 0.38−0.48 (m, 2H), 0.82−0.96 (m, 1H), 1.22−1.37 (m, 2H), 1.55−1.80 (m, 3H), 2.97−3.04 (m, 2H), 3.19−3.36 (m, 4H), 3.89−3.99 (m, 2H), 6.91 (dd, J = 7.0, 8.8 Hz, 1H), 7.52 (d, J = 7.0 Hz, 1H), 7.62 (d, J = 8.8 Hz, 1H). 3-[(Cyclopropylmethyl)[(tetrahydro-2H-pyran-4-yl)methyl]amino]-7-[2,6-dimethoxy-4-(methoxymethyl)phenyl]pyrazolo[1,5-a]pyridine-2-carbonitrile (45). To a sulution of 44 (103 mg, 0.24 mmol) in toluene (2 mL) and EtOH (1 mL) were added [2,6dimethoxy-4-(methoxymethyl)phenyl)]boronic acid (103 mg, 0.45 mmol), Pd(PPh3)4 (27 mg, 0.024 mmol) and 1 M Na2CO3 aqueous solution (0.47 mL, 0.47 mmol), and the mixture was stirred at 90 °C for 3 h under a nitrogen stream. The reaction mixture was cooled and water was added. The mixture was extracted with EtOAc, and the organic layer was washed with brine, dried over MgSO4, filtered, and concentrated in vacuo. The residue was purified by column chromatography on NH silica gel (n-heptane:EtOAc = 2:1), followed by normal silica gel (n-hexane:EtOAc = 1:1) to afford 45 (70 mg, 60%) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ −0.07−0.03 (m, 2H), 0.35−0.44 (m, 2H), 0.85−0.97 (m, 1H), 1.22−1.36 (m, 2H), 1.59−1.73 (m, 1H), 1.73−1.83 (m, 2H), 2.95−3.02 (m, 2H), 3.17− 3.24 (m, 2H), 3.26−3.36 (m, 2H), 3.48 (s, 3H), 3.73 (s, 6H), 3.89− 3.98 (m, 2H), 4.53 (s, 2H), 6.87 (d, J = 6.8 Hz, 1H), 6.68 (s, 2H), 7.18 (dd, J = 6.8, 8.8 Hz, 1H), 7.55 (d, J = 8.8 Hz, 1H). 13C NMR (150 MHz, CDCl3) δ 158.7, 142.6, 136.9, 134.7, 129.9, 123.1, 122.4, 117.6, 116.3, 115.1, 109.2, 103.2, 74.8, 67.9, 62.0, 60.6, 58.5, 56.0, 34.1, 31.3, 9.8, 3.5. HRMS calcd for (C28H34N4O4) [M + H]+ 491.2653; found 491.2660. N-(Cyclopropylmethyl)-7-[2,6-dimethoxy-4(methoxymethyl)phenyl]-2-ethyl-N-[(tetrahydro-2H-pyran-4yl)methyl]pyrazolo[1,5-a]pyridin-3-amine 4-methylbenzenesulfonate (46). To a solution of 24r (1.6 g, 3.2 mmol) in EtOAc (25 mL) was added p-toluenesulfonic acid monohydrate (617 mg, 3.2 mmol), and the mixture was stirred at room temperature. The precipitate was filtered out to afford a crude product (2.18 g), which was recrystallized from a mixed solvent of tert-butyl methyl ether (640 mL) and EtOAc (770 mL) to afford 46 (1.9 g, 89%) as a white crystal. mp 201 °C. 1H NMR (400 MHz, C5D5N) δ −0.03−0.07 (m, 2H), 0.30−0.40 (m, 2H), 0.70−0.82 (m, 1H), 1.22 (dddd, J = 5, 12, 13, 13 Hz, 2H), 1.32 (t, J = 8 Hz, 3H), 1.56 (ttt, J = 4, 7, 13 Hz, 1H), 1.68 (br d, J = 13 Hz, 2H), 2.16 (s, 3H), 2.79 (d, J = 7 Hz, 2H), 2.87 (q, J = 8 Hz, 2H), 2.95 (d, J = 7 Hz, 2H), 3.22 (ddd, J = 5, 12, 12 Hz, 2H), 3.38 (s, 3H), 3.52 (s, 6H), 3.95 (br dd, J = 2, 12 Hz, 2H), 4.50 (s, 2H), 6.85 (s, 2H), 6.88 (dd, J = 1, 7 Hz, 1H), 7.18 (d, J = 8 Hz, 2H), 7.21 (dd, J = 7, 9 Hz, 1H), 7.63 (dd, J = 1, 9 Hz, 1H), 8.37 (d, J = 8 Hz, 2H). 13C 5267

dx.doi.org/10.1021/jm300259r | J. Med. Chem. 2012, 55, 5255−5269

Journal of Medicinal Chemistry

Article

was a white open-top box (light box; 20 × 15 × 20 cm3 high). A black acrylic tunnel (7 × 10 × 4.5 cm3 high) separated the dark box from the light box. In order to record the behavior of animals, the front- and back-sides of the light box (20 × 20 cm2) were made of clear acrylic glass. The light intensity on floor of the light box was fixed to 150 lx. At the beginning of the experiment, a mouse was placed in the dark box. Its behavior was recorded on video tape over a 5 min period and the following two parameters were noted by an observer who was unaware of the treatment each animal had received: (a) the time spent in the light box, and (b) the number of line crosses between the dark tunnel and the light box. A mouse whose four paws were in the light box was considered as being in the light box. Data are expressed as the mean ± standard error of the mean (S.E.M.). Differences between the vehicle control and 46 or 1 treated groups were evaluated by one-way analysis of variance, followed by Dunnett multiple comparison test. A value of p < 0.05 (two-sided) was considered statistically significant. Determination of In Vitro Hepatic Clearance. The in vitro hepatic clearance data were determined by depletion in human liver microsomes. Pooled human liver microsomes (n = 150) were purchased from BD (MA, USA). The stability assay was conducted by using 0.3 μmol/L substrate and 0.1 mg/mL microsomal protein in which the final concentration of organic solvent was 0.03% DMSO. Incubation was conducted at 37 °C for 0 and 15 min by adding the NADPH generating system. After the incubation, the microsomal matrix was deproteinized by adding acetonitrile/methanol containing the internal standard. After centrifugation, the resulting supernatant was analyzed by LC/MS/MS. Mouse Pharmacokinetic Study. Pharmacokinetic parameters were estimated in male fasted CD1 (ICR) mice (Charles River Japan Inc., Kanagawa, Japan) after intravenous (IV) (3 mg/kg, 10 mL/kg) and oral (PO) (10 mg/kg, 10 mL/kg) administration. Dosing solution was prepared in DMSO/5% glucose with 1/250 volume of 5 mol/L HCl (1:19 for IV and 1:9 for PO). Blood samples were collected from the vena cava at 0.083 (5 min; for IV), 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h after dosing (n = 4 at each time point). Plasma was separated by centrifugation and stored in a frozen state until analysis. Plasma concentrations were measured by an HPLC−fluorescence method. A plasma sample (50 μL), 50 μL of internal standard solution, and 500 μL of 0.1 mol/L phosphate buffer (pH 7.4) were mixed, followed by the addition of 2 mL of isopropyl alcohol/n-hexane (3:97, v/v). Samples were shaken for 10 min and centrifuged. The organic layer was transferred to a new tube to which 500 μL of 0.2 mol/L HCl was added. This tube was again shaken for 10 min and centrifuged. After centrifugation, the resulting water layer (50 μL) was injected into the HPLC column. Chromatography was performed in the reversed-phase mode with a Mightysil RP-8 GP (4.6 mm i.d. × 150 mm, Kanto Chemical Co., Ltd., Tokyo, Japan) using gradient elution with two mobile phases (A and B) at 1 mL/min. Mobile phase A consisted of distilled water/acetonitrile/60% HClO4/NaClO4 (800:200:1:5, v/v/v/ w), and mobile phase B consisted of distilled water/acetonitrile/60% HClO4/NaClO4 (200:800:1:5, v/v/v/w). Compound 46 and the internal standard were detected using a fluorescence detector (Ex: 305 nm, Em: 410 nm). Kinetic Solubility Assay. Water solubility was determined as follows: Sample solutions were prepared by dilution of 2.5 μL of 10 mM DMSO stock solution with 250 μL of Dulbecco’s phosphate buffered saline in a 96-well filter plate. The plate was shaken for 15 min at room temperature to allow the compounds to equilibrate. The sample solutions were filtered into another 96-well plate by vacuum. Standard solutions were prepared by dilution of 2.5 μL of 10 mM stock DMSO solution with 250 μL of DMSO in a 96-well plate. The filtrated sample solutions and standard solutions were analyzed by HPLC to determine the solubility.

■

k, 24g, 24m−o, 27a−b, 30b−g, 33. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*Tel.: +81 (0)29 847 7395; Fax: +81 (0)29 847 4952; e-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors thank Dr. Kodo Shikata for performing the mouse light/dark tests; Mami Gomibuchi, for HRMS; Eri Ena and Yumi Yokoyama, for NMR analysis; and Dr. Taro Terauchi, Dr. Yuichi Suzuki, Dr. Noritaka Kitazawa, Dr. Luis Castro, and Dr. Teiji Kimura for their assistance in the preparation of the manuscript.

■

ABBREVIATIONS USED CRF, corticotropin-releasing factor; hCLint, intrinsic clearance in human liver microsomes; THP, tetrahydropyranyl

■

REFERENCES

(1) Vale, W.; Spiess, J.; Rivier, C.; Rivier, J. Characterization of a 41residue ovine hypothalamic peptide that stimulates secretion of corticotropin and beta-endorphin. Science 1981, 213, 1394−1397. (2) Owens, M. J.; Nemeroff, C. B. Physiology and pharmacology of corticotropin-releasing factor. Pharmacol. Rev. 1991, 43, 425−473. (3) Spiess, J.; Dautzenberg, F. M.; Sydow, S.; Hauger, R. L.; Ruhmann, A.; Blank, T.; Radulovic, J. Molecular properties of the CRF Receptor. Trends Endocrinol. Metab. 1998, 9, 140−145. (4) Kostich, W. A.; Chen, A.; Sperle, K.; Largent, B. L. Molecular identification and analysis of a novel human corticotropin releasing factor (CRF) receptor: the CRF2γ receptor. Mol. Endocrinol. 1998, 12, 1077−1085. (5) Chen, R.; Lewis, K. A.; Perrin, M. H.; Vale, W. W. Expression cloning of a human corticotropin-releasing-factor receptor. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 8967−8971. (6) Perrin, M. H.; Grace, C. R.; Riek, R.; Vale, W. W. The ThreeDimensional Structure of the N-Terminal Domain of CorticotropinReleasing Factor Receptors. Sushi Domains and the B1 Family of G Protein−Coupled Receptors. Ann. N.Y. Acad. Sci. 2006, 1070, 105− 119. (7) Smith, G. W.; Aubry, J.-M.; Dellu, F.; Contrarino, A.; Bilezekijian, L. M.; Gold, L. H.; Chen, R.; archuk, Y.; Hauser, C.; Bentley, C. A.; Sawchenko, P. E.; Koob, G. F.; Vale, W.; Lee, K.-F. Corticotropin releasing factor receptor 1-deficient mice display decreased anxiety, impaired stress response, and aberrant neuroendocrine development. Neuron 1998, 20, 1093−1102. (8) Timpl, P.; Spanagel, R.; Sillabar, I.; Kresse, A.; Reul, J. M.; Stalla, G. K.; Blanquet, V.; Steckler, T.; Holsboer, F.; Wurst, W. Impaired stress response and reduced anxiety in mice lacking a functional corticotropin-releasing hormone receptor 1. Nat. Genet. 1998, 19, 162−166. (9) Dunn, A. J.; Berridge, C. W. Physiological and behavioral responses to corticotropin-releasing factor administration: is CRF a mediator of anxiety or stress responses? Brain Res. Rev. 1990, 15, 71− 100. (10) Nemeroff, C. B.; Widerlov, E.; Bissette, G.; Wallens, H.; Karlsson, I.; Eklund, K.; Kilts, C. D.; Loosen, P. T.; Vale, W. Elevated concentrations of CSF corticotropin-releasing factor-like immunoreactivity in depressed patients. Science 1984, 226, 1342−1344. (11) Arborelius, L.; Owens, M. J.; Plotsky, P. M.; Nemeroff, C. B. The role of corticotropin-releasing factor in depression and anxiety disorders. J. Endocrinol. 1999, 160, 1−12.

ASSOCIATED CONTENT

S Supporting Information *

Table of HPLC purity data, and experimental procedures for the preparation of compounds 30b−g and the intermediates 12−14, 16−18, 22, 23, 25, 26, 31, 32, 34, used for 24c, 24 h− 5268

dx.doi.org/10.1021/jm300259r | J. Med. Chem. 2012, 55, 5255−5269

Journal of Medicinal Chemistry

Article

(12) Zorrilla, E. P.; Koob, G. F. Progress in corticotropin-releasing factor-1 antagonist development. Drug Discovery Today 2010, 15, 371−383. (13) Ising, M.; Zimmermann, U. S.; Kuenzel, H. E.; Uhr, M.; Foster, A. C.; Learned-Coughlin, S. M.; Holsboer, F.; Grigoriadis, D. E. HighAffinity CRF1 Receptor Antagonist NBI-34041: Preclinical and Clinical Data Suggest Safety and Efficacy in Attenuating Elevated Stress Response. Neuropsychopharmacology 2007, 32, 1941−1949. (14) Heinrichs, S. C.; De Souza, E. B.; Schulteis, G.; Lapsansky, J. L.; Grigoriadis, D. E. Brain penetrance, receptor occupancy and antistress in vivo efficacy of a small molecule corticotropin releasing factor type I receptor selective antagonist. Neuropsychopharmacology 2002, 27, 194−202. (15) Gutman, D. A.; Owens, M. J.; Skelton, K. H.; Thrivikraman, K. V.; Nemeroff, C. B. The Corticotropin-Releasing Factor1 Receptor Antagonist R121919 Attenuates the Behavioral and Endocrine Responses to Stress. J. Pharmacol. Exp. Ther. 2003, 304, 874−880. (16) Mansbach, R. S.; Brooks, E. N.; Chen, Y. L. Antidepressant-like effects of CP-154,526, a selective CRF1 receptor antagonist. Eur. J. Pharmacol. 1997, 323, 21−26. (17) Arborelius, L.; Skelton, K. H.; Thrivikraman, K. V.; Plotsky, P. M.; Schulz, D. W.; Owens, M. J. Chronic Administration of the Selective Corticotropin-Releasing Factor 1 Receptor Antagonist CP154,526: Behavioral, Endocrine and Neurochemical Effects in the Rat. J. Pharmacol. Exp. Ther. 2000, 294, 588−597. (18) Schulz, D. W.; Mansbach, R. S.; Sprouse, J.; Braselton, J. P.; Collins, J.; Corman, M.; Dunaiskis, A.; Faraci, S.; chmidt, A. W.; Seeger, T.; Seymour, P.; Tingley, F. D.; Winston, E. N., III; Chen, , Y.; Heym, J. CP-154526, a potent and selective nonpeptide antagonist of corticotropin releasing factor receptors. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 10477−10482. (19) Li, Y.-W.; Hill, G.; Wong, H.; Kelly, N.; Ward, K.; Pierdomenico, M.; Ren, S.; Gilligan, P.; Grossman, S.; Trainor, G.; aub, R.; McElroy, J.; Zaczek, R. Receptor Occupancy of Nonpeptide Corticotropin-Releasing Factor 1 Antagonist DMP696: Correlation with Drug Exposure and Anxiolyic Efficacy. J. Pharmacol. Exp. Ther. 2003, 305, 86−96. (20) Chen, C.; Grigoriadis, D. E. NBI 30775 (R121919), an Orally Active Antagonist of the Corticotropin-releasing Factor (CRF) Type-1 Receptor for the Treatment of Anxiety and Depression. Drug Dev. Res. 2005, 65, 216−226. (21) Zobel, A. W.; Nickel, T.; Künzel, H. E.; Ackl, N.; Sonntag, A.; Ising, M.; Holsboer, F. Effects of high-affinity corticotropin-releasing hormone receptor 1 antagonist R121919 in major depression: the first 20 patients treated. J. Psychiatric Res. 2000, 34, 171−181. (22) Binneman, B.; Feltner, D.; Kolluri, S.; Shi, Y.; Qiu, R.; Stiger, T. A 6-week randomized, placebo-controlled trial of CP-316,311 (a selective CRH1 antagonist) in the treatment of major depression. Amer. J. Psychiatry 2008, 165, 617−620. (23) Chen, C.; Wilcoxen, K. M.; Huang, C. Q.; Xie, Y.-F.; McCarthy, J. R.; Webb, T. R.; Zhu, Y.-F.; Saunders, J.; Liu, X.-J.; Chen, T.-K.; Bozigian, H.; Grigoriadis, D. E. Design of 2,5-dimethyl-3- (6-dimethyl4-methylpyridin-3-yl)-7-dipropylaminopyrazolo[1,5-a] pyrimidine (NBI 30775/R121919) and structure-activity relationships of a series of potent and orally active corticotropin-releasing factor receptor antagonists. J. Med. Chem. 2004, 47, 4787−4798. (24) Chen, Y. L.; Mansbach, R. S.; Winter, S. M.; Brooks, E.; Collins, J.; Corman, M. L.; Dunaiskis, A. R.; Faraci, W. S.; allaschun, R. J.; Schmidt, A.; Schultz, D. W. Synthesis and oral efficacy of a 4(butylethylamino)pyrrolo[2,3-d]pyrimidine: a centrally active corticotropin-releasing factor1 receptor antagonist. J. Med. Chem. 1997, 40, 1749−1754. (25) He, L.; Gilligan, P. J.; Zaczek, R.; Fitzgerald, L. W.; McElroy, J.; Shen, H.-S. L.; Saye, J. A.; Kalin, N. H.; Shelton, S.; Christ, D.; Trainor, G.; Hartig, P. 4-(1,3-Dimethoxyprop-2-ylamino)-2,7-dimethyl-8-(2,4dichlorophenyl)-pyrazolo[1,5-a]-1,3,5-triazine: a potent, orally bioavailable CRF1 receptor antagonist. J. Med. Chem. 2000, 43, 449−456. (26) Chen, Y. L.; Braselton, J.; Forman, J.; Gallaschun, R. J.; Mansbach, R.; Schmidt, A. W.; Seeger, T. F.; Sprouse, J. F.; Tingley, F.

D.; Winston, E., III; Schulz, D. W. Synthesis and SAR of 2-aryloxy-4alkoxy-pyridines as potent orally active corticotropinreleasing factor 1 receptor antagonists. J. Med. Chem. 2008, 51, 1377−1384. (27) Gilligan, P. J.; Robertson, D. W.; Zaczek, R. Corticotropin Releasing Factor (CRF) Receptor Modulators: Progress and Opportunities for New Therapeutic Agents. J. Med. Chem. 2000, 43, 1641−1660. (28) Tamura, Y.; Minamikawa, J.; Sumoto, K.; Fujii, S.; Ikeda, M. Synthesis and some properties of O-acyl and O-nitrophenylhydroxylamines. J. Org. Chem. 1973, 38, 1239−1241. (29) Tsuchiya, T.; Sashida, H.; Konoshita, A. Thermal Rearrangements of Cyclic Amine Ylides. III. Intramolecular Cyclization of 2Ethynylpyridine N-Imides to 3-Azaindolizine Derivatives. Chem. Pharm. Bull. 1983, 31, 4568−5572. (30) Ochi, H.; Miyasaka, T.; Kanada, K.; Arakawa, K. Studies of Heterocyclic Compounds. VIII. Synthesis and Tautomerism of 2Hydroxypyrazolo[1,5-a]pyridine. Bull. Chem. Soc. Jpn. 1976, 49, 1980− 1984. (31) Fujito, H.; Tominaga, Y.; Matsuda, Y.; Kobayashi, G. Reaction of Pyridinium and Isoquinolinium N-imines with Ketenethioacetals. Heterocycles 1977, 6, 379−382. (32) Chen, R.; Lewis, A. K.; Perrin, H. M; Vale, W. Expression cloning of a human corticotropin-releasing-factor receptor. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 8967−8971. (33) ClogP was calculated by software of Daylight Chemical Information Systems, Inc. (34) Stepan, A. F.; Karki, K.; McDonald, W. S.; Dorff, P. H.; Dutra, J. K.; DiRico, K. J.; Won, A.; Subramanyam, C.; Efremov, I. V.; O’Donnell, C. J.; Nolan, C. E; Becker, S. L.; Pustilnik, L. R.; Sneed, B.; Sun, H.; Lu, Y.; Robshaw, A. E.; Riddell, D.; O’Sullivan, T. J.; Sibley, E.; Capetta, S.; Atchison, K.; Hallgren, A. J.; Miller, E.; Wood, A.; Obach, R. S. Metabolism-Directed Design of Oxetane-Containing Arylsulfonamide Derivatives as γ-Secretase Inhibitors. J. Med. Chem. 2011, 54, 7772−7783. (35) Sugaya, Y.; Yoshiba, T.; Kajima, T.; Ishihama, Y. Development of Solubility Screening Methods in Drug Discovery. YAKUGAKU ZASSHI 2002, 122, 237−246. (36) Okuyama, S.; Chaki, S.; Kawashima, N.; Suzuki, Y.; Ogawa, A.; Nakazato, A.; Kumagai, T.; Okubo, T.; Tomisawa, K. Receptor binding, behavioral, and electrophysiological profiles of nonpeptide corticotropin-releasing factor subtype 1 receptor antagonists CRA1000 and CRA1001. J. Pharm. Exp. Ther. 1999, 289, 926−935. (37) Belzung, C.; Misslin, R.; Vogel, E. Behavioural effects of the benzodiazepine receptor partial agonist RO 16−6028 in mice. Psychopharmacology 1989, 97, 388−391.

5269

dx.doi.org/10.1021/jm300259r | J. Med. Chem. 2012, 55, 5255−5269